Rna-targeting system

ABSTRACT

The invention provides for systems, methods, and compositions for targeting RNA. In particular, the invention provides a non-naturally occurring or engineered RNA-targeting system comprising an RNA-targeting Cas protein and at least one RNA-targeting guide RNA, wherein said RNA-targeting guide RNA is capable of hybridizing with a target RNA in a cell.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of International patentapplication Serial No. PCT/US2015/067151 filed Dec. 21, 2015 andpublished as PCT Publication No. WO2016/106236 on Jun. 30, 2016 andwhich claims the benefit of and priority from U.S. application Ser. No.62/096,324, filed Dec. 23, 2014, U.S. application Ser. No. 62/098,059,filed Dec. 30, 2014, U.S. application Ser. No. 62/181,641, filed Jun.18, 2015 and U.S. application Ser. No. 62/181,667, filed Jun. 18, 2015.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberMH100706, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

The foregoing application, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 23, 2016, isnamed 47627.99.2009_SL.txt and is 12 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as perturbation of gene transcripts or RNA editing, thatmay use vector systems related to Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new RNA targeting technologies that enable diverseapplications, such as RNA knockdown, translational regulation ortranscript splicing, trafficking, and visualization. Simple and scalabletools to study and manipulate RNA lag significantly behind their DNAcounterparts. Existing RNA interference technologies, which enablecleavage or inhibition of desired transcripts, have significantoff-target effects and remain challenging engineering targets due totheir key role in endogenous processes. One of the key limitations inRNA engineering has been the lack of RNA-binding domains that can beeasily retargeted. The MS2 RNA-binding domain, for example, recognizesan invariable 21 nt RNA sequence, requiring genomic modification to taga desired transcript. While pumilio homology domains possess modularrepeats similar to TAL effectors, with each protein module recognizing aseparate RNA base, they recognize only 8 nucleotides of RNA and haveproved difficult to reprogram in practice. Transcriptional activationand repression can also be achieved through Cas9-mediated recruitment ofprotein effectors. However, such modes of transcriptional control onlyallow coarse gene regulation via DNA promoter or enhancer regions ofinterest. Hence, there remains a need for RNA engineering technologiesthat are affordable, easy to set up, scalable, and amenable to targetingmultiple RNA targets.

Sampson et al. (2013, Nature 497: 254-258) describe a naturalCRISPR-Cas-mediated system that represses an endogenous transcript inFrancisella novicida. This system uses the Cas protein Cas9 ofFrancisella novicida, a unique, small, CRISPR/Cas-associated RNA(scaRNA), and trans-activating crRNA (tracrRNA) to repress an endogenoustranscript encoding a bacterial lipoprotein. The system has not beentransferred yet into heterologous cells.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for RNA targeting with a wide array of applications. Thisinvention addresses this need and provides related advantages. Thetargeting system described herein has dual functionality as it cantarget both DNA and RNA depending on the targeting guide selected.Aspects of the RNA-targeting system described herein may be modifiedaccordingly for use as a DNA-targeting system. The RNA-targeting systemalso referred to as RNA-targeting CRISPR/Cas or the CRISPR-CasRNA-targeting system of the present application which is based on an RT(RNA-targeting) type II Cas protein (e.g. the Cas9 from the bacterialpathogen Francisella novicida) does not require the generation ofcustomized proteins to target specific RNA sequences but rather a singleCas enzyme can be programmed by an RNA molecule to recognize a specificRNA target, in other words the Cas enzyme can be recruited to a specificRNA target using said RNA molecule. Adding the novel RNA-targetingsystem of the present application to the repertoire of RNA targetingtechnologies may transform the study and perturbation or editing of RNAthrough direct RNA detection, analysis and manipulation. To utilize theRNA-targeting system of the present application effectively for RNAtargeting without deleterious effects, it is critical to understandaspects of engineering and optimization of these RNA targeting tools.

The RNA-targeting systems, the vector systems, the vectors and thecompositions described herein may be used in various RNA-targetingapplications, altering or modifying synthesis of a gene product, such asa protein, RNA cleavage, RNA editing, RNA splicing; trafficking oftarget RNA, tracing of target RNA, isolation of target RNA,visualization of target RNA, etc.

Accordingly, in a further aspect, the invention provides a method foraltering, modifying or modulating synthesis of a gene product, inparticular a protein. The said method may comprise introducing into acell containing and expressing an RNA molecule encoding the geneproduct, in particular the protein, an engineered or non-naturallyoccurring CRISPR-Cas RNA-targeting system as taught herein comprising anRNA-targeting Cas protein (such as eg. the Cas9 from the bacterialpathogen Francisella novicida) and an RNA-targeting guide RNA thattargets the RNA molecule encoding the gene product, wherein theRNA-targeting guide RNA comprises an scaRNA sequence and a tracrRNAsequence, which scaRNA sequence and tracrRNA sequence are capable of atleast partially hybridizing, whereby synthesis of the gene product, inparticular the protein, is altered; and, wherein the RNA-targeting Casprotein and the RNA-targeting guide RNA do not naturally occur together.In a preferred embodiment, said method comprises introducing into a cellcontaining an RNA molecule encoding said gene product, in particularsaid protein, a vector system comprising one or more vectors comprising:

a) a first regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting guide RNA, wherein said RNA-targetingguide RNA is capable of hybridizing with said target RNA, wherein saidRNA-targeting guide RNA comprises:(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein said scaRNA and said tracrRNA are capable of at least partiallyhybridizing, andb) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein,wherein components (a) and (b) are located on the same or differentvectors of the system; whereby synthesis of said gene product, inparticular said protein, is modulated. In a preferred embodiment thecell is a eukaryotic cell, and in more preferred embodiment theeukaryotic cell is a mammalian cell, and in an even more preferredembodiment the mammalian cell is a human cell. In an embodiment of theinvention, the synthesis of the gene product is decreased or suppressed.In a further embodiment of the invention, the synthesis of the geneproduct is suppressed by knock-down of the RNA molecule encoding thegene product. In another embodiment of the invention, the synthesis ofthe gene product is increased. In an embodiment of the invention, thesynthesis of the gene product, in particular the protein, is modulatedby editing the RNA molecule encoding the gene product, e.g. the protein.In an embodiment of the invention, the synthesis of the gene product, inparticular the protein, is modulated by splicing the RNA moleculeencoding the gene product, e.g. the protein.

In an aspect, the invention provides a method for targeting RNA. Saidmethod may comprise introducing into a cell containing a target RNA, avector system as taught herein comprising one or more vectorscomprising:

a) a first regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting guide RNA, wherein said RNA-targetingguide RNA is capable of hybridizing with said target RNA, wherein saidRNA-targeting guide RNA comprises:(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein said scaRNA and said tracrRNA are capable of at least partiallyhybridizing, andb) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein,wherein components (a) and (b) are located on the same or differentvectors of the system; whereby said RNA-targeting guide RNA directs saidRNA-targeting Cas protein to said target RNA.

Also disclosed herein is a method for tracing a target RNA into a cellcomprising:

introducing into the cell containing the target RNA a non-naturallyoccurring or engineered vector system comprising one or more vectorscomprising:

a) a first regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting guide RNA, wherein the RNA-targetingguide RNA is capable of hybridizing with the target RNA, wherein theRNA-targeting guide RNA comprises:(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein the scaRNA and the tracrRNA are capable of at least partiallyhybridizing, andb) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein, wherein theRNA-targeting Cas protein is fused to an identification tag, whereincomponents (a) and (b) are located on the same or different vectors ofthe system, and

visualizing the identification tag.

Further disclosed herein is a method of isolating a target RNA from acell comprising:

introducing into a cell containing the target RNA a non-naturallyoccurring or engineered vector system comprising one or more vectorscomprising:

a) a first regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting guide RNA, wherein the RNA-targetingguide RNA is capable of hybridizing with the target RNA, wherein theRNA-targeting guide RNA comprises:(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein the scaRNA and the tracrRNA are capable of at least partiallyhybridizing, andb) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein, wherein theRNA-targeting Cas protein is fused to a tag or associated with a bead,wherein components (a) and (b) are located on the same or differentvectors of the system, and

separating the target RNA from the cell using the tag or the bead.

In particular, an object of the current invention is to further enhancethe specificity of RNA targeting (RT) type II Cas system of the presentinvention given individual guide RNAs through thermodynamic tuning ofthe binding specificity of the guide RNA to target RNA.

In one aspect, the invention provides an engineered, non-naturallyoccurring CRISPR-Cas RNA-targeting system comprising an RT type II Casprotein and an RNA-targeting guide RNA that targets an RNA molecule fortranscribing a gene product in a cell, whereby the guide RNA targets theRNA molecule transcribing the gene product and the Cas protein binds toand optionally cleaves the RNA molecule encoding the gene product,whereby expression of the gene product is altered or modulated; and,wherein the Cas protein and the guide RNA do not naturally occurtogether. The invention comprehends the RNA-targeting guide RNA whichoptionally comprises a guide sequence fused to a tracr sequence. In anembodiment of the invention the Cas protein is a type II CRISPR-Casprotein having RNA targeting properties and in a preferred embodimentthe Cas protein is a Cas9 protein e.g. from the bacterial pathogenFrancisella novicida or orthologs or homologs or fragments thereofhaving similar RNA targeting properties. The invention furthercomprehends the Cas protein to be used according to the invention beingcodon optimized for expression in a eukaryotic cell. In a preferredembodiment the eukaryotic cell is a mammalian cell and in a morepreferred embodiment the mammalian cell is a human cell. In a furtherembodiment of the invention, the expression of the gene product isdecreased.

In another aspect, the invention provides an engineered, non-naturallyoccurring vector system comprising one or more vectors comprising afirst regulatory element operably linked to an RNA-targeting guide RNAthat targets an RNA molecule encoding a gene product and a secondregulatory element operably linked to an RNA-targeting Cas protein.Components (a) and (b) may be located on same or different vectors ofthe system. The RNA-targeting guide RNA targets the RNA moleculeencoding the gene product in a cell and the RNA-targeting Cas proteinbinds to and optionally cleaves the RNA molecule encoding the geneproduct, whereby expression of the gene product is altered; and theRNA-targeting Cas protein and the RNA-targeting guide RNA do notnaturally occur together. In particular embodiments, the inventioncomprehends the RNA-targeting guide RNA to comprise a small CRISPR/Cassystem associated RNA (scaRNA) sequence fused to a tracr sequence. Inparticular embodiments, the RNA-targeting guide RNA comprises a guide(sequence directing recognition of the target RNA), a tracr mate and atracr RNA sequence, wherein the tracr mate hybridizes with the tracrRNA. In an embodiment of the invention the RNA-targeting Cas protein isan RT type II CRISPR-Cas protein and in a preferred embodiment the Casprotein is a Cas9 protein such as FnCas9 protein. The invention furthercomprehends the Cas protein being codon optimized for expression in aeukaryotic cell. In a preferred embodiment the eukaryotic cell is amammalian cell and in a more preferred embodiment the mammalian cell isa human cell. In a further embodiment of the invention, the expressionof the gene product is decreased.

In one aspect, the invention provides a vector system comprising one ormore vectors. In some embodiments, the RNA-targeting system comprises:(a) a first regulatory element operably linked to a tracr mate sequenceand one or more insertion sites for inserting one or more guidesequences upstream of the tracr mate sequence, wherein when expressed,the guide sequence directs sequence-specific binding of an RNA-targetingCRISPR complex to a target sequence in a eukaryotic cell, wherein theRNA-targeting CRISPR complex comprises an RNA-targeting type II Casenzyme complexed with (1) the guide sequence that is hybridized to thetarget sequence, and (2) the tracr mate sequence that is hybridized tothe tracr sequence; and (b) a second regulatory element operably linkedto an enzyme-coding sequence encoding said RNA-targeting type II Casenzyme comprising a nuclear localization sequence; wherein components(a) and (b) are located on the same or different vectors of the system.In some embodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of an RNA-targeting CRISPR complex to adifferent target sequence in a eukaryotic cell. In some embodiments, thesystem comprises the tracr sequence under the control of a thirdregulatory element, such as a polymerase III promoter. In someembodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%,90%, 95%, or 99%, of sequence complementarity along the length of thetracr mate sequence when optimally aligned. Determining optimalalignment is within the purview of one of skill in the art. For example,there are publically and commercially available alignment algorithms andprograms such as, but not limited to, ClustalW, Smith-Waterman inmatlab, Bowtie, Geneious, Biopython and SeqMan. In some embodiments, theRNA-targeting CRISPR complex comprises one or more nuclear localizationsequences of sufficient strength to drive accumulation of saidRNA-targeting CRISPR complex in a detectable amount in the nucleus of aeukaryotic cell. Without wishing to be bound by theory, it is believedthat a nuclear localization sequence is not necessary for RNA-targetingCRISPR complex activity in eukaryotes, but that including such sequencesmay enhance activity of the system especially as to targeting RNAmolecules in the nucleus. In some embodiments, the RNA-targeting enzymeis a type II Cas system enzyme. In some embodiments, the CRISPR enzymeis a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Francisellanovicida Cas9, (Fn) and may include mutated Cas9 derived from theseorganisms. The enzyme may be an RT (Fn)Cas9 homolog or ortholog. In someembodiments, the CRISPR enzyme is codon-optimized for expression in aeukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavageof the target sequence. In some embodiments, the CRISPR enzyme lacks DNAstrand cleavage activity. In some embodiments, the first regulatoryelement is a polymerase III promoter. In some embodiments, the secondregulatory element is a polymerase II promoter. In some embodiments, theguide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, orbetween 10-30, or between 15-25, or between 15-20 nucleotides in length.In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Common expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the 3-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

In one aspect, the invention provides a vector comprising a regulatoryelement operably linked to an enzyme-coding sequence encoding a Type IIRT CasCRISPR enzyme. In some embodiments, said regulatory element drivestranscription of the CRISPR enzyme in a eukaryotic cell such that saidRNA-targeting enzyme accumulates in a detectable amount in the nucleusof the eukaryotic cell. In some embodiments, the regulatory element is apolymerase II promoter. In some embodiments, the RNA-targeting enzyme isa type II CRISPR system enzyme. In some embodiments, the CRISPR enzymeis a Cas9 enzyme. In some embodiments, the Cas9 enzyme is Francisellanovicida Cas9 (FnCas9), and may include mutated Cas9 derived from theseorganisms. In some embodiments, the RNA targeting enzyme iscodon-optimized for expression in a eukaryotic cell. In someembodiments, the RNA targeting enzyme directs cleavage of one or twostrands at the location of the target sequence. In some embodiments, theRNA targeting enzyme lacks DNA strand cleavage activity.

The RNA-targeting enzyme may be an RNA targeting Cas9 homolog orortholog, particularly of a Francisella novicida Cas9 enzyme. In someembodiments, the CRISPR enzyme lacks the ability to cleave one or morestrands of a target sequence to which it binds.

In one aspect, the invention provides a eukaryotic host cell comprising(a) a first regulatory element operably linked to an RNA-targeting guidesequence comprising one or more insertion sites for inserting one ormore guide sequences, wherein when expressed, the guide sequence directssequence-specific binding of an RNA-targeting complex to an RNA targetsequence in a eukaryotic cell, wherein the RNA-targeting complexcomprises an RNA-targeting type II Cas enzyme complexed with (1) theRNA-targeting guide RNA. In further particular embodiments, theRNA-targeting complex may comprise (1) the guide sequence of theRNA-targeting guide RNA that is hybridized to the target sequence, and(2) the tracr mate sequence that is hybridized to the tracr sequence;and/or (b) a second regulatory element operably linked to anenzyme-coding sequence encoding said Type II RT Cas enzyme. In someembodiments, the host cell comprises components (a) and (b). In someembodiments, component (a), component (b), or components (a) and (b) arestably integrated into a genome of the host eukaryotic cell. In someembodiments, component (a) further comprises the tracr sequencedownstream of the tracr mate sequence under the control of the firstregulatory element. In some embodiments, component (a) further comprisestwo or more guide sequences operably linked to the first regulatoryelement, wherein when expressed, each of the two or more guide sequencesdirect sequence specific binding of a CRISPR complex to a different RNAtarget sequence in a eukaryotic cell. In some embodiments, theeukaryotic host cell further comprises a third regulatory element, suchas a polymerase III promoter, operably linked to said tracr sequence. Insome embodiments, the tracr sequence exhibits at least 50%, 60%, 70%,80%, 90%, 95%, or 99% of sequence complementarity along the length ofthe tracr mate sequence when optimally aligned. In some embodiments, theRT-type II Cas enzyme comprises one or more nuclear localizationsequences of sufficient strength to drive accumulation of said CRISPRenzyme in a detectable amount of a eukaryotic cell. The CRISPR enzymeused according to the invention is a type II RT Cas CRISPR systemenzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In someembodiments, the Cas9 enzyme is a Francisella novicida Type II RT Cas9,and may include mutated Cas9 derived from these organisms. The enzymemay be an RT Cas9 homolog or ortholog. In some embodiments, the CRISPRenzyme is codon-optimized for expression in a eukaryotic cell. In someembodiments, the RNA-targeting CRISPR enzyme lacks DNA strand cleavageactivity. In some embodiments, the first regulatory element is apolymerase III promoter. In some embodiments, the second regulatoryelement is a polymerase II promoter. In some embodiments, the guidesequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between10-30, or between 15-25, or between 15-20 nucleotides in length. In anaspect, the invention provides a non-human eukaryotic organism;preferably a multicellular eukaryotic organism, comprising a eukaryotichost cell according to any of the described embodiments. In otheraspects, the invention provides a eukaryotic organism; preferably amulticellular eukaryotic organism, comprising a eukaryotic host cellaccording to any of the described embodiments. The organism in someembodiments of these aspects may be an animal; for example a mammal.Also, the organism may be an arthropod such as an insect. The organismalso may be a plant. Further, the organism may be a fungus.

With respect to use of the CRISPR-Cas system generally, mention is madeof the documents, including patent applications, patents, and patentpublications cited throughout this disclosure as embodiments of theinvention can be used as in those documents. CRISPR-Cas system(s) (e.g.,single or multiplexed) can be used in conjunction with recent advancesin crop genomics. Such CRISPR-Cas system(s) can be used to performefficient and cost effective plant gene or genome interrogation orediting or manipulation—for instance, for rapid investigation and/orselection and/or interrogations and/or comparison and/or manipulationsand/or transformation of plant genes or genomes; e.g., to create,identify, develop, optimize, or confer trait(s) or characteristic(s) toplant(s) or to transform a plant genome. There can accordingly beimproved production of plants, new plants with new combinations oftraits or characteristics or new plants with enhanced traits. SuchCRISPR-Cas system(s) can be used with regard to plants in Site-DirectedIntegration (SDI) or Gene Editing (GE) or any Near Reverse Breeding(NRB) or Reverse Breeding (RB) techniques. With respect to use of theCRISPR-Cas system in plants, mention is made of the University ofArizona website “CRISPR-PLANT” (http://www.genome.arizona.edu/crispr/)(supported by Penn State and AGI). Embodiments of the invention can beused in genome editing in plants or where RNAi or similar genome editingtechniques have been used previously; see, e.g., Nekrasov, “Plant genomeediting made easy: targeted mutagenesis in model and crop plants usingthe CRISPR/Cas system,” Plant Methods 2013, 9:39 (doi:10.1186/1746-4811-9-39); Brooks. “Efficient gene editing in tomato inthe first generation using the CRISPR/Cas9 system,” Plant PhysiologySeptember 2014 pp 114.247577; Shan, “Targeted genome modification ofcrop plants using a CRISPR-Cas system,” Nature Biotechnology 31, 686-688(2013); Feng, “Efficient genome editing in plants using a CRISPR/Cassystem,” Cell Research (2013) 23:1229-1232. doi:10.1038/cr.2013.114;published online 20 Aug. 2013; Xie, “RNA-guided genome editing in plantsusing a CRISPR-Cas system,” Mol Plant. 2013 November; 6(6):1975-83. doi:10.1093/mp/sst119. Epub 2013 Aug. 17; Xu, “Gene targeting using theAgrobacterium tumefaciens-mediated CRISPR-Cas system in rice,” Rice2014, 7:5 (2014), Zhou et al., “Exploiting SNPs for biallelic CRISPRmutations in the outcrossing woody perennial Populus reveals4-coumarate: CoA ligase specificity and Redundancy,” New Phytologist(2015) (Forum) 1-4 (available online only at newphytologist.com);Caliando et al, “Targeted DNA degradation using a CRISPR device stablycarried in the host genome, NATURE COMMUNICATIONS 6:6989, DOI:10.1038/ncomms7989, www.nature.com/naturecommunications DOI:10.1038/ncomms7989; U.S. Pat. No. 6,603,061—Agrobacterium-Mediated PlantTransformation Method; U.S. Pat. No. 7,868,149—Plant Genome Sequencesand Uses Thereof and US 2009/0100536—Transgenic Plants with EnhancedAgronomic Traits, all the contents and disclosure of each of which areherein incorporated by reference in their entirety. In the practice ofthe invention, the contents and disclosure of Morrell et al “Cropgenomics: advances and applications,” Nat Rev Genet. 2011 Dec. 29;13(2):85-96; each of which is incorporated by reference herein includingas to how herein embodiments may be used as to plants. Accordingly,reference herein to animal cells may also apply, mutatis mutandis, toplant cells unless otherwise apparent.

In one aspect, the invention provides a kit comprising one or more ofthe components described herein. In some embodiments, the kit comprisesa vector system and instructions for using the kit. In some embodiments,the vector system comprises one or more of an RNA-targeting type II Casenzyme and an RNA targeting guide RNA, wherein the RNA-targeting guideRNA comprises (i) a small CRISPR/Cas system associated RNA (scaRNA)sequence, and (ii) a trans-activating CRISPR/Cas system RNA (tracrRNA)sequence, wherein said scaRNA and said tracrRNA are capable of at leastpartially hybridizing. In particular embodiments, the kit comprises avector system comprising (a) a first regulatory element operably linkedto a tracr mate sequence and one or more insertion sites for insertingone or more guide sequences upstream of the tracr mate sequence, whereinwhen expressed, the guide sequence directs sequence-specific binding ofan RNA-targeting CRISPR complex to an RNA target sequence in aeukaryotic cell, wherein the RNA-targeting CRISPR complex comprises aType II RT Cas CRISPR enzyme complexed with (1) the guide sequence thatis hybridized to the target sequence, and (2) the tracr mate sequencethat is hybridized to the tracr sequence; and/or (b) a second regulatoryelement operably linked to an enzyme-coding sequence encoding saidCRISPR enzyme comprising a nuclear localization sequence. In someembodiments, the kit comprises components (a) and (b) located on thesame or different vectors of the system. In some embodiments, component(a) further comprises the tracr sequence downstream of the tracr matesequence under the control of the first regulatory element. In someembodiments, component (a) further comprises two or more guide sequencesoperably linked to the first regulatory element, wherein when expressed,each of the two or more guide sequences direct sequence specific bindingof an RNA-targeting CRISPR complex to a different target RNA sequence ina eukaryotic cell. In some embodiments, the system further comprises athird regulatory element, such as a polymerase III promoter, operablylinked to said tracr sequence. In some embodiments, the tracr sequenceexhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequencecomplementarity along the length of the tracr mate sequence whenoptimally aligned. In some embodiments, the RNA-targeting enzymecomprises one or more nuclear localization sequences of sufficientstrength to drive accumulation of said enzyme in a detectable amount inthe nucleus of a eukaryotic cell. In some embodiments, the RNA-targetingenzyme is a type II CRISPR system enzyme. In some embodiments, theRNA-targeting enzyme is a Cas9 enzyme. In some embodiments, the Cas9enzyme is Francisella novicida Type II RT Cas9, and may include mutatedCas9 derived from this organism. The enzyme may be a Cas9 homolog orortholog. In some embodiments, the CRISPR enzyme is codon-optimized forexpression in a eukaryotic cell. In some embodiments, the RNA-targetingenzyme lacks DNA strand cleavage activity. In some embodiments theRNA-targeting enzyme lacks RNA cleavage activity. In some embodiments,the first regulatory element is a polymerase III promoter. In someembodiments, the second regulatory element is a polymerase II promoter.In some embodiments, the guide sequence is at least 15, 16, 17, 18, 19,20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20nucleotides in length.

In one aspect, the invention provides a method of modulating synthesisof protein in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR complex to bind to the target RNA to effectcleavage of said target RNA thereby modifying the transcription of aprotein translated from said RNA, wherein the RNA-targeting complexcomprises an RNA-targeting type II Cas enzyme complexed with anRNA-targeting guide RNA hybridized to a target sequence. In particularembodiments, the RNA-targeting complex comprises an RNA-targeting typeII Cas enzyme complexed with a guide sequence hybridized to a targetsequence within said target RNA, wherein said guide sequence is linkedto a tracr mate sequence which in turn hybridizes to a tracr sequence.In some embodiments, said RNA-targeting enzyme cleaves said target RNA.In some embodiments, said cleavage results in decreased transcription ofa target gene. In some embodiments, the method further comprises editingsaid RNA, wherein said editing results in a mutation comprising aninsertion, deletion, or substitution of one or more nucleotides of saidtarget RNA. In some embodiments, said editing results in one or moreamino acid changes in a protein translated from the RNA target sequence.In some embodiments, the method further comprises delivering one or morevectors to said eukaryotic cell, wherein the one or more vectors driveexpression of one or more of: the RNA-targeting type II Cas enzyme andthe RNA-targeting guide RNA. In further embodiments the vectors driveexpression of the RNA-targeting type II Cas enzyme the guide sequencelinked to the tracr mate sequence, and the tracr sequence. In someembodiments, said vectors are delivered to the eukaryotic cell in asubject. In some embodiments, said modifying takes place in saideukaryotic cell in a cell culture. In some embodiments, the methodfurther comprises isolating said eukaryotic cell from a subject prior tosaid modifying. In some embodiments, the method further comprisesreturning said eukaryotic cell and/or cells derived therefrom to saidsubject.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing an RNA-targeting complex to bind to the RNAcorresponding to said polynucleotide such that said binding results inincreased or decreased expression of said polynucleotide; wherein theRNA-targeting complex comprises an RNA-targeting type II Cas enzymecomplexed with an RNA-targeting guide RNA hybridized to a target RNAsequence corresponding to said polynucleotide. In particularembodiments, the RNA-targeting complex comprises an RNA-targeting typeII Cas enzyme complexed with a guide sequence hybridized to a targetsequence within said polynucleotide, wherein said guide sequence islinked to a tracr mate sequence which in turn hybridizes to a tracrsequence. In some embodiments, the method further comprises deliveringone or more vectors to said eukaryotic cells, wherein the one or morevectors drive expression of one or more of: the RNA-targeting enzyme andthe RNA-targeting guide RNA. More particularly the one or more vectorsdrive expression of one or more of, the guide sequence linked to thetracr mate sequence, and the tracr sequence.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a modified transcript of a disease gene. Insome embodiments, a disease gene is any gene associated an increase inthe risk of having or developing a disease. In some embodiments, themethod comprises (a) introducing one or more vectors into a eukaryoticcell, wherein the one or more vectors drive expression of one or moreof: an RNA-targeting type II Cas enzyme and an RNA-targeting guide RNA.In particular embodiments, the one or more vectors drive expression ofone or more of an RNA targeting enzyme, a guide sequence linked to atracr mate sequence, and a tracr sequence; the method may furthercomprise (b) allowing an RNA-targeting complex to bind to a target RNAto effect cleavage of or binding to the target RNA of said diseasegene), wherein the RNA-targeting complex comprises the RNA-targetingenzyme complexed with the RNA-targeting guide RNA hybridized to thetarget sequence. In particular embodiments, the RNA-targeting complexcomprises the RNA-targeting enzyme complexed with (1) the guide sequencethat is hybridized to the target sequence within the targetpolynucleotide, and (2) the tracr mate sequence that is hybridized tothe tracr sequence, thereby generating a model eukaryotic cellcomprising a mutated disease transcript. In some embodiments, saidcleavage or binding results in decreased translation of a target RNA. Insome embodiments, this results in a mutation comprising an insertion,deletion, or substitution of one or more nucleotides of said target RNA.In some embodiments, said mutation results in one or more amino acidchanges in a protein transcription from an RNA comprising the targetsequence.

In one aspect, the invention provides a method for developing abiologically active agent that modulates a cell signaling eventassociated with a disease RNA. In some embodiments, a disease RNA is anyRNA associated an increase in the risk of having or developing adisease. In some embodiments, the method comprises (a) contacting a testcompound with a model cell of any one of the described embodiments; and(b) detecting a change in a readout that is indicative of a reduction oran augmentation of a cell signaling event associated with said mutationin said disease RNA, thereby developing said biologically active agentthat modulates said cell signaling event associated with said diseaseRNA.

In one aspect, the invention provides a recombinant polynucleotidecomprising a sequence corresponding to an RNA-targeting guide RNA. Inparticular embodiments, the RNA-targeting guide RNA comprises a guidesequence upstream of a tracr mate sequence, wherein the guide sequencewhen expressed directs sequence-specific binding of an RNA-targetingcomplex to a corresponding target sequence present in a eukaryotic cell.In some embodiments, the target sequence is a viral sequence present ina eukaryotic cell. In some embodiments, the target RNA sequence is aproto-oncogene RNA or an oncogene RNA.

In one aspect the invention provides for a method of selecting one ormore cell(s) by introducing one or more modifications in the RNA in theone or more cell(s), the method comprising: introducing one or morevectors into the cell(s), wherein the one or more vectors driveexpression of one or more of: a CRISPR enzyme, a guide sequence linkedto a tracr mate sequence, a tracr sequence, and an editing template;wherein the editing template comprises the one or more mutations thatabolish CRISPR enzyme cleavage; allowing homologous recombination of theediting template with the target polynucleotide in the cell(s) to beselected; allowing a CRISPR complex to bind to a target polynucleotideto effect cleavage of the target polynucleotide within said gene,wherein the CRISPR complex comprises the CRISPR enzyme complexed with(1) the guide sequence that is hybridized to the target sequence withinthe target polynucleotide, and (2) the tracr mate sequence that ishybridized to the tracr sequence, wherein binding of the CRISPR complexto the target polynucleotide induces cell death, thereby allowing one ormore cell(s) in which one or more mutations have been introduced to beselected. In a preferred embodiment, the CRISPR enzyme is Cas9. Inanother aspect of the invention the cell to be selected may be aeukaryotic cell. Aspects of the invention allow for selection ofspecific cells without requiring a selection marker or a two-stepprocess that may include a counter-selection system. The cell(s) may beprokaryotic or eukaryotic cells.

With respect to mutations of the CRISPR enzyme, when the enzyme is notSpCas9, mutations may be made at any or all residues corresponding topositions 10, 762, 840, 854, 863 and/or 986 of Francisella novicida(which may be ascertained for instance by standard sequence comparisontools). In particular, any or all of the following mutations arepreferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; aswell as conservative substitution for any of the replacement amino acidsis also envisaged. In an aspect the invention provides as to any or eachor all embodiments herein-discussed wherein the CRISPR enzyme comprisesat least one or more, or at least two or more mutations, wherein the atleast one or more mutation or the at least two or more mutations is asto D10, E762, H840, N854, N863, or D986 according to SpCas9 protein,e.g., D10A, E762A, H840A, N854A, N863A and/or D986A as to SpCas9, orN580 according to SaCas9, e.g., N580A as to SaCas9, or any correspondingmutation(s) in a Cas9 of an ortholog to Sp or Sa, or the CRISPR enzymecomprises at least one mutation wherein at least H840 or N863A as to SpCas9 or N580A as to Sa Cas9 is mutated; e.g., wherein the CRISPR enzymecomprises H840A, or D10A and H840A, or D10A and N863A, according toSpCas9 protein, or any corresponding mutation(s) in a Cas9 of anortholog to Sp protein or Sa protein.

In a further aspect, the invention involves a computer-assisted methodfor identifying or designing potential compounds to fit within or bindto a CRISPR-Type II RT Cas system or a functional portion thereof orvice versa (a computer-assisted method for identifying or designingpotential CRISPR-Type II RT systems or a functional portion thereof forbinding to desired compounds) or a computer-assisted method foridentifying or designing potential CRISPR-Type II RT Cas systems (e.g.,with regard to predicting areas of the CRISPR-Type II RT Cas system tobe able to be manipulated—for instance, based on crystal structure dataor based on data of FnCas9 orthologs, or with respect to where afunctional group such as an activator or repressor can be attached tothe CRISPR-Type II RT Cas system, or as to Type II RT Cas (such asFnCas9) truncations or as to designing nickases), said methodcomprising:

using a computer system, e.g., a programmed computer comprising aprocessor, a data storage system, an input device, and an output device,the steps of:

(a) inputting into the programmed computer through said input devicedata comprising the three-dimensional co-ordinates of a subset of theatoms from or pertaining to the CRISPR-Type II RT Cas crystal structure,e.g., in the CRISPR-Type II RT Cas system binding domain oralternatively or additionally in domains that vary based on varianceamong such Cas orthologs or as to such Cass or as to nickases or as tofunctional groups, optionally with structural information fromCRISPR-Type II RT Cas system complex(es), thereby generating a data set;

(b) comparing, using said processor, said data set to a computerdatabase of structures stored in said computer data storage system,e.g., structures of compounds that bind or putatively bind or that aredesired to bind to a CRISPR-Type II RT Cas system or as to Type II RTCas (such as FNCas9) orthologs (e.g., as Cas9s or as to domains orregions that vary amongst Cas9 orthologs) or as to the CRISPR-Type II RTCas crystal structure or as to nickases or as to functional groups;

(c) selecting from said database, using computer methods,structure(s)—e.g., CRISPR-Type II RT Cas structures that may bind todesired structures, desired structures that may bind to certainCRISPR-Type II RT Cas structures, portions of the CRISPR-Type II RT Cassystem that may be manipulated, e.g., based on data from other portionsof the CRISPR-Type II RT Cas crystal structure and/or from Type II RTCas (eg. FnCas9) orthologs, truncated versions, novel nickases orparticular functional groups, or positions for attaching functionalgroups or functional-group-CRISPR-Type II RT Cas systems;

(d) constructing, using computer methods, a model of the selectedstructure(s); and

(e) outputting to said output device the selected structure(s);

and optionally synthesizing one or more of the selected structure(s);

and further optionally testing said synthesized selected structure(s) asor in a CRISPR-Type II RT Cas system;

or, said method comprising: providing the co-ordinates of at least twoatoms of the CRISPR-Type II RT Cas (eg. FN Cas9 crystal structure, e.g.,at least two atoms of the herein Crystal Structure Table of theCRISPR-Cas9 crystal structure or co-ordinates of at least a sub-domainof the CRISPR-Type II RT Cas crystal structure (“selectedco-ordinates”), providing the structure of a candidate comprising abinding molecule or of portions of the CRISPR-Type II RT Cas system thatmay be manipulated, e.g., based on data from other portions of theCRISPR-Type II RT Cas crystal structure and/or from Type II RT Casorthologs, or the structure of functional groups, and fitting thestructure of the candidate to the selected co-ordinates, to therebyobtain product data comprising CRISPR-Type II RT Cas structures that maybind to desired structures, desired structures that may bind to certainCRISPR-Type II RT Cas structures, portions of the CRISPR-Type II RT Cassystem that may be manipulated, truncated Type II RT Cas's, novelnickases, or particular functional groups, or positions for attachingfunctional groups or functional-group-CRISPR-Type II RT Cas systems,with output thereof; and optionally synthesizing compound(s) from saidproduct data and further optionally comprising testing said synthesizedcompound(s) as or in a CRISPR-Type II RT Cas system.

The testing can comprise analyzing the CRISPR-Type II RT Cas systemresulting from said synthesized selected structure(s), e.g., withrespect to binding, or performing a desired function.

The output in the foregoing methods can comprise data transmission,e.g., transmission of information via telecommunication, telephone,video conference, mass communication, e.g., presentation such as acomputer presentation (eg POWERPOINT), internet, email, documentarycommunication such as a computer program (eg WORD) document and thelike. Accordingly, the invention also comprehends computer readablemedia containing: atomic co-ordinate data according to theherein-referenced Crystal Structure, said data defining the threedimensional structure of CRISPR-Type II RT Cas or at least onesub-domain thereof, or structure factor data for CRISPR-Type II RT Cas,said structure factor data being derivable from the atomic co-ordinatedata of herein-referenced Crystal Structure. The computer readable mediacan also contain any data of the foregoing methods. The inventionfurther comprehends methods a computer system for generating orperforming rational design as in the foregoing methods containingeither: atomic co-ordinate data according to herein-referenced CrystalStructure, said data defining the three dimensional structure ofCRISPR-Type II RT Cas or at least one sub-domain thereof, or structurefactor data for CRISPR-Type II RT Cas, said structure factor data beingderivable from the atomic co-ordinate data of herein-referenced CrystalStructure. The invention further comprehends a method of doing businesscomprising providing to a user the computer system or the media or thethree dimensional structure of CRISPR-Type II RT Cas or at least onesub-domain thereof, or structure factor data for CRISPR-Type II RT Cas,said structure set forth in and said structure factor data beingderivable from the atomic co-ordinate data of herein-referenced CrystalStructure, or the herein computer media or a herein data transmission.

A “binding site” or an “active site” comprises or consists essentiallyof or consists of a site (such as an atom, a functional group of anamino acid residue or a plurality of such atoms and/or groups) in abinding cavity or region, which may bind to a compound such as a nucleicacid molecule, which is/are involved in binding.

By “fitting”, is meant determining by automatic, or semi-automaticmeans, interactions between one or more atoms of a candidate moleculeand at least one atom of a structure of the invention, and calculatingthe extent to which such interactions are stable. Interactions includeattraction and repulsion, brought about by charge, steric considerationsand the like. Various computer-based methods for fitting are describedfurther

By “root mean square (or rms) deviation”, we mean the square root of thearithmetic mean of the squares of the deviations from the mean.

By a “computer system”, is meant the hardware means, software means anddata storage means used to analyze atomic coordinate data. The minimumhardware means of the computer-based systems of the present inventiontypically comprises a central processing unit (CPU), input means, outputmeans and data storage means. Desirably a display or monitor is providedto visualize structure data. The data storage means may be RAM or meansfor accessing computer readable media of the invention. Examples of suchsystems are computer and tablet devices running Unix, Windows or Appleoperating systems.

By “computer readable media”, is meant any medium or media, which can beread and accessed directly or indirectly by a computer e.g., so that themedia is suitable for use in the above-mentioned computer system. Suchmedia include, but are not limited to: magnetic storage media such asfloppy discs, hard disc storage medium and magnetic tape; opticalstorage media such as optical discs or CD-ROM; electrical storage mediasuch as RAM and ROM; thumb drive devices; cloud storage devices andhybrids of these categories such as magnetic/optical storage media.

In particular embodiments of the invention, the conformationalvariations in the crystal structures of the CRISPR-Type II RT Cas systemor of components of the CRISPR-Type II RT Cas provide important andcritical information about the flexibility or movement of proteinstructure regions relative to nucleotide (RNA or DNA) structure regionsthat may be important for CRISPR-Type II RT system function. Thestructural information provided for Type II RT Cas (Cas9 and e.g.,Francisella novicida Cas9 as the CRISPR enzyme in the presentapplication may be used to further engineer and optimize the CRISPR-TypeII RT Cas system and this may be extrapolated to interrogatestructure-function relationships in other RNA-targeting CRISPR enzymesystems as well, e.g. other Type II CRISPR enzyme systems.

The invention comprehends optimized functional CRISPR-Type II RT Casenzyme systems. In particular the CRISPR enzyme comprises one or moremutations that converts it to an RNA binding protein to which functionaldomains exhibiting a function of interest may be recruited or appendedor inserted or attached. In certain embodiments, the CRISPR-Type II RTCas enzyme may comprises one or more mutations. In some embodiments, theRNA-targeting CRISPR enzyme has one or more mutations in a catalyticdomain, wherein when transcribed, the RNA-targeting guide sequencedirects sequence-specific binding of an RNA-targeting CRISPR complex tothe target sequence, and wherein the enzyme further comprises afunctional domain.

The structural information provided herein allows for interrogation ofRNA-targeting RNA guide interaction with the target RNA and the Type IIRT Cas enzyme (e.g., FnCas9) permitting engineering or alteration ofRNA-targeting structure to optimize functionality of the entireRNA-targeting (CRISPR-Cas) system. For example, the RNA-targeting RNAguide may be extended, without colliding with the Cas protein by theinsertion of adaptor proteins that can bind to RNA. These adaptorproteins can further recruit effector proteins or fusions which compriseone or more functional domains.

The compositions of the invention encompass a non-naturally occurring orengineered composition that may comprise an RNA-targeting guide RNARt-gRNA) comprising a guide sequence capable of hybridizing to an RNAtarget sequence of interest in a cell and an RNA-targeting Cas enzyme,wherein the RNA-targeting Cas enzyme comprises two or more mutations,such that the enzyme has altered or diminished nuclease activitycompared with the wild type enzyme. In particular embodiments, theRNA-targeting guide RNA is modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins, and wherein theadaptor protein further recruits one or more heterologous functionaldomains. An aspect of the invention encompasses methods of modifying anRNA of interest to change RNA translation in a cell by introducing intothe cell any of the compositions described herein.

An aspect of the invention is that the above elements are comprised in asingle composition or comprised in individual compositions. Thesecompositions may advantageously be applied to a host to elicit afunctional effect on the RNA level.

In general, the RNA-targeting guide RNA (Rt-gRNA) is modified in amanner that provides specific binding sites (e.g., aptamers) for adapterproteins comprising one or more functional domains (e.g., via fusionprotein) to bind to. The modified Rt-gRNA are modified such that oncethe Rt-gRNA forms a CRISPR complex (i.e. RNA-targeting enzyme binding toRt-gRNA and target) the adapter proteins bind and, the functional domainon the adapter protein is positioned in a spatial orientation which isadvantageous for the attributed function to be effective. The skilledperson will understand that modifications to the Rt-gRNA which allow forbinding of the adapter+functional domain but not proper positioning ofthe adapter+functional domain (e.g., due to steric hinderance within thethree dimensional structure of the CRISPR complex) are modificationswhich are not intended.

As explained herein the functional domains may be, for example, one ormore domains from the group comprising, consisting essentially of, orconsisting of methylase activity, demethylase activity, translationactivation activity, translation repression activity, histonemodification activity, RNA cleavage activity, DNA cleavage activity,nucleic acid binding activity, and molecular switches (e.g., lightinducible). In some cases it is advantageous that additionally at leastone NLS is provided. In some instances, it is advantageous to positionthe NLS at the N terminus. When more than one functional domain isincluded, the functional domains may be the same or different.

The RNA targeting guide RNA or Rt-gRNA may be designed to includemultiple binding recognition sites (e.g., aptamers) specific to the sameor different adapter protein. The Rt-gRNA may be designed to bind to thepromoter region −1000−+1 nucleic acids upstream of the transcriptionstart site (i.e. TSS), preferably −200 nucleic acids. This positioningimproves functional domains which affect gene activation (e.g.,transcription activators) or gene inhibition (e.g., transcriptionrepressors). The modified Rt-gRNA may be one or more modified Rt-gRNAstargeted to one or more target loci (e.g., at least 1 sgRNA, at least 2sgRNA, at least 5 sgRNA, at least 10 sgRNA, at least 20 sgRNA, at least30 sg RNA, at least 50 sgRNA) comprised in a composition.

Where the RNA targeting enzyme has natural RNA nuclease activity, the RTenzyme may be modified to have diminished nuclease activity e.g.,nuclease inactivation of at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, or 100% as compared with the wild type enzyme;or to put in another way, a Cas9 enzyme having advantageously about 0%of the nuclease activity of the non-mutated or wild type Cas9 enzyme orCRISPR enzyme, or no more than about 3% or about 5% or about 10% of thenuclease activity of the non-mutated or wild type Type II RT Cas enzyme,e.g. of the non-mutated or wild type Francisella novicida Cas9 enzyme orCRISPR enzyme. This is possible by introducing mutations into thenuclease domains of the Francisella novicida Cas9 and orthologs thereof.

The inactivated Type II RT Cas CRISPR enzyme may have associated (e.g.,via fusion protein) one or more functional domains, including forexample, one or more domains from the group comprising, consistingessentially of, or consisting of methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, RNA cleavage activity, DNA cleavage activity, nucleic acidbinding activity, and molecular switches (e.g., light inducible).Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In the event thatFok1 is provided, it is advantageous that multiple Fok1 functionaldomains are provided to allow for a functional dimer and that sgRNAs aredesigned to provide proper spacing for functional use (Fok1) asspecifically described in Tsai et al. Nature Biotechnology, Vol. 32,Number 6, June 2014). The adaptor protein may utilize known linkers toattach such functional domains. In some cases it is advantageous thatadditionally at least one NLS is provided. In some instances, it isadvantageous to position the NLS at the N terminus. When more than onefunctional domain is included, the functional domains may be the same ordifferent.

In general, the positioning of the one or more functional domain on theinactivated Type II RT Cas CRISPR enzyme is one which allows for correctspatial orientation for the functional domain to affect the target withthe attributed functional effect. For example, if the functional domainis a transcription activator (e.g., VP64 or p65), the transcriptionactivator is placed in a spatial orientation which allows it to affectthe transcription of the target. Likewise, a transcription repressorwill be advantageously positioned to affect the transcription of thetarget, and a nuclease (e.g., Fok1) will be advantageously positioned tocleave or partially cleave the target. This may include positions otherthan the N-/C-terminus of the CRISPR enzyme.

Due to crystal structure experiments on SpCas9, the Applicant hasidentified that positioning the functional domain in the Rec1 domain,the Rec2 domain, the HNH domain, or the PI domain of the Francisellanovicida Cas9 protein or any ortholog corresponding to these domains isadvantageous. Positioning of the functional domains to the Rec1 domainor the Rec2 domain, of the protein or any ortholog corresponding tothese domains, in some instances may be preferred. Positioning of thefunctional domains to the Rec1 domain at position 553, Rec1 domain at575, the Rec2 domain at any position of 175-306 or replacement thereof,the HNH domain at any position of 715-901 or replacement thereof, or thePI domain at position 1153 of the Francisella novicida protein or anyortholog corresponding to these domains, in some instances may bepreferred. Fok1 functional domain may be attached at the N terminus.When more than one functional domain is included, the functional domainsmay be the same or different.

The adaptor protein may be any number of proteins that binds to anaptamer or recognition site introduced into the modified Rt-gRNA andwhich allows proper positioning of one or more functional domains, oncethe Rt-gRNA has been incorporated into the RNA-targeting complex, toaffect the target or other polynucleotides or proteins associatedtherewith with the attributed function. As explained in detail in thisapplication such may be coat proteins, preferably bacteriophage coatproteins. The functional domains associated with such adaptor proteins(e.g., in the form of fusion protein) may include, for example, one ormore domains from the group comprising, consisting essentially of, orconsisting of methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity, DNA cleavage activity, nucleic acid binding activity, andmolecular switches (e.g., light inducible). Preferred domains are Fok1,VP64, P65, HSF1, MyoD1. In the event that the functional domain is atranscription activator or transcription repressor it is advantageousthat additionally at least an NLS is provided and preferably at the Nterminus. When more than one functional domain is included, thefunctional domains may be the same or different. The adaptor protein mayutilize known linkers to attach such functional domains.

Thus, the modified Rt-gRNA, the (inactivated) Type II RT Cas CRISPRenzyme (with or without functional domains), and the binding proteinwith one or more functional domains, may each individually be comprisedin a composition and administered to a host individually orcollectively. Alternatively, these components may be provided in asingle composition for administration to a host. Administration to ahost may be performed via viral vectors known to the skilled person ordescribed herein for delivery to a host (e.g., lentiviral vector,adenoviral vector, AAV vector). As explained herein, use of differentselection markers and concentration of Rt-gRNA (e.g., dependent onwhether multiple Rt-gRNAs are used) may be advantageous for eliciting animproved effect.

The compositions may be applied in a wide variety of methods forscreening in libraries in cells and functional modeling in vivo (e.g.,gene activation of lincRNA and identification of function;gain-of-function modeling; loss-of-function modeling; the use thecompositions of the invention to establish cell lines and transgenicanimals for optimization and screening purposes).

The current invention comprehends the use of the compositions of thecurrent invention to establish and utilize conditional or inducibleCRISPR transgenic cell/animals. (See, e.g., Platt et al., Cell (2014),159(2): 440-455, or PCT patent publications cited herein, such as WO2014/093622 (PCT/US2013/074667), which are not believed prior to thepresent invention or application). For example, the target cellcomprises Type II RT Cas CRISRP enzyme (e.g., FnCas9) conditionally orinducibly (e.g., in the form of Cre dependent constructs) and/or theadapter protein conditionally or inducibly and, on expression of avector introduced into the target cell, the vector expresses that whichinduces or gives rise to the condition of CRISRP enzyme (e.g., FnCas9)expression and/or adaptor expression in the target cell. By applying theteaching and compositions of the current invention with the known methodof creating a CRISPR complex, inducible RNA events affected byfunctional domains are also an aspect of the current invention. One mereexample of this is the creation of a CRISPR RNA knock-in/conditionaltransgenic animal (e.g., mouse comprising e.g., aLox-Stop-polyA-Lox(LSL) cassette) and subsequent delivery of one or morecompositions providing one or more modified Rt-gRNA (e.g., −200nucleotides to TSS of a target gene of interest for gene activationpurposes) as described herein (e.g., modified Rt-gRNA with one or moreaptamers recognized by coat proteins, e.g., MS2), one or more adapterproteins as described herein (MS2 binding protein linked to one or moreVP64) and means for inducing the conditional animal (e.g., Crerecombinase for rendering Type II RT Cas expression inducible).Alternatively, the adaptor protein may be provided as a conditional orinducible element with a conditional or inducible CRISPR enzyme toprovide an effective model for screening purposes, which advantageouslyonly requires minimal design and administration of specific sgRNAs for abroad number of applications.

In any of the vector systems, vectors, compositions or methods describedherein one or more amino acid residues of the RNA-targeting Cas proteinmay be modified. Modification may comprise mutation of one or more, ortwo or more, or three or more, amino acid residues of the RNA-targetingCas protein. The one or more, or two or more, or three or more,mutations may be in one or more catalytically active domains of theRNA-targeting Cas protein.

In any such mutants the RNA-targeting Cas protein may have reduced orabolished nuclease activity compared with an RNA-targeting Cas proteinlacking said mutations. Any of such one or more, or two or more, orthree or more, mutations may be in a catalytically active domain of theRNA-targeting Cas protein comprising a RuvCI, RuvCII or RuvCIII domain.

In any of the vector systems, vectors, compositions or methods describedherein the RNA-targeting Cas protein may comprise one or moreheterologous functional domains.

The one or more heterologous functional domains may comprise one or morenuclear localization signal (NLS) domains, or at least two or more NLSdomains.

The one or more heterologous functional domains may comprise one or moretranscriptional activation domains, such as VP64. The one or moreheterologous functional domains may comprise one or more transcriptionalrepression domains, such as a KRAB domain or a SID domain. The one ormore heterologous functional domains may comprise one or more nucleasedomains, such as Fok1. The one or more heterologous functional domainsmay have one or more of the following activities: methylase activity,demethylase activity, transcription activation activity, transcriptionrepression activity, transcription release factor activity, histonemodification activity, nuclease activity, single-strand RNA cleavageactivity, double-strand RNA cleavage activity, single-strand DNAcleavage activity, double-strand DNA cleavage activity and nucleic acidbinding activity.

The one or more heterologous functional domains may be at or near theamino-terminus of the RNA-targeting Cas protein and/or at or near thecarboxy-terminus of the RNA-targeting Cas protein.

The one or more heterologous functional domains may be fused to theRNA-targeting Cas protein or tethered to the RNA-targeting Cas proteinor linked to the RNA-targeting Cas protein by a linker moiety.

In any of the vector systems, vectors, compositions or methods describedherein the RNA-targeting Cas protein may comprise an RNA-targeting Casprotein from an organism from a genus comprising Streptococcus,Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta,Lactobacillus, Eubacterium or Corynebacter. The RNA-targeting Casprotein may comprise a chimeric RNA-targeting Cas protein comprising afirst fragment from a first RNA-targeting Cas protein ortholog and asecond fragment from a second RNA-targeting Cas protein ortholog, andwherein the first and second RNA-targeting Cas protein orthologs aredifferent and wherein at least one of the first and second orthologs isFrancisella novicida. At least one of the first and second RNA-targetingCas protein orthologs may comprise an RNA-targeting Cas protein from anorganism comprising Streptococcus, Campylobacter, Nitratifractor,Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter,Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter.

In any of the vector systems, vectors, compositions or methods describedherein the tracrRNA may comprise one or more protein-binding RNAaptamers. The scaRNA may comprise one or more protein-binding RNAaptamers. The one or more aptamers are capable of binding abacteriophage coat protein. The bacteriophage coat protein may beselected from the group comprising Qβ, F2, GA, fr, JP501, MS2, M12, R17,BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19,AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. Preferably, thebacteriophage coat protein is MS2.

In any of the vector systems, vectors, compositions or methods describedherein the tracrRNA may be 30 or more, 40 or more or 50 or morenucleotides in length.

In any of the vector systems, vectors, compositions or methods describedherein the RNA-targeting Cas protein may be further modified (e.g. bythe introduction of one or more amino acid mutations, bytruncation/deletion and/or insertion of one or more specific amino acidsor amino acid sequences) to alter PAM specificity.

In any of the vector systems, vectors, compositions or methods describedherein multiple Rt-gRNAs may be delivered, wherein each Rt-gRNAs isspecific for a different target RNA whereby there is multiplexing.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 provides a photograph representing a gel shift assay showingFnCas9 binding to tracr RNA, sgRNA, and FTN_1103.

FIG. 2A-2B provides a graph representing the processing states of scaRNAas determined by RNA sequencing (SEQ ID NO: 35). FIG. 2B provides agraph representing the processing states of tracrRNA as determined byRNA sequencing (SEQ ID NO: 36).

FIG. 3 provides a photograph of a Western blot showing FnCas9 expressionin E. coli.

FIG. 4 provides a graph representing the expression of the bacteriallipoprotein FTN_1103 in E. coli cells transformed with plasmidscomprising the F. novicida CRISPR/Cas locus along with the FTN-1103 generelative to control (E. coli comprising no FnCas9 and no F. novicidaCRISPR/Cas locus); Black: 1:10 dilution; light grey: 1:100 dilution;dark grey: 1:1000 dilution.

FIG. 5 provides a schematic representation of a method forcharacterizing potential enzymatic maturation of scaRNA and tracrRNA.

FIG. 6 provides a schematic representation of a method for screening forretargeted fSca guide sequences.

FIG. 7 provides a schematic representation of a method for studying thespecificity and efficiency of fSca guide sequences.

FIG. 8 provides a phylogenic tree with plots of species with more than 1putative tracrRNA/scaRNA.

FIG. 9A-9C provides scaRNA sequences. FIG. 9A provides the sequence ofscaRNA from Sampson et al. (2013, Nature 497: 254-258) (SEQ ID NOS30-32, respectively, in order of appearance). FIG. 9B provides thesequence of Applicants' scaRNA from RNA sequencing (SEQ ID NO: 33) andFIG. 9C provides RNA Seq reads on ScaRNA (SEQ ID NO: 34).

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

In general, the CRISPR-Cas or CRISPR system is as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667) and referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)enzyme, including sequences encoding or delivering a Cas enzyme (DNAand/or RNA-targeting) enzyme, a tracr (trans-activating CRISPR) sequence(e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence(encompassing a “direct repeat” and a tracrRNA-processed partial directrepeat in the context of an endogenous CRISPR system), a guide sequence(also referred to as a “spacer” in the context of an endogenous CRISPRsystem), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guideCas9, e.g., CRISPR RNA and transactivating (tracr) RNA or a single guideRNA (sgRNA) (chimeric RNA)), a small RNA-targeting CRISPR/Cas systemassociated sequence (“sca-RNA”) or other sequences and transcripts froma CRISPR locus. In general, a CRISPR system is characterized by elementsthat promote the formation of a CRISPR complex at the site of a targetsequence (also referred to as a protospacer in the context of anendogenous CRISPR system). In the context of formation of a CRISPRcomplex, “target sequence” refers to a sequence to which a guidesequence is designed to have complementarity, where hybridizationbetween a target sequence and a guide sequence promotes the formation ofa CRISPR complex. A target sequence may comprise any polynucleotide,such as DNA or RNA polynucleotides. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In someembodiments, direct repeats may be identified in silico by searching forrepetitive motifs that fulfill any or all of the following criteria: 1.found in a 2 Kb window of genomic sequence flanking the type II CRISPRlocus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. Insome embodiments, 2 of these criteria may be used, for instance 1 and 2,2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

The term “RNA-targeting system” refers collectively to transcripts andother elements involved in the expression of or directing the activityof RNA-targeting CRISPR-associated (“Cas”) genes, including sequencesencoding an RNA-targeting Cas protein and an RNA-targeting guide RNAcomprising an RNA-targeting small CRISPR/Cas system associated RNA(scaRNA) sequence and an RNA-targeting trans-activating CRISPR/Cassystem RNA (tracrRNA) sequence, or other sequences and transcripts froman RNA-targeting CRISPR locus. In general, an RNA-targeting system ischaracterized by elements that promote the formation of an RNA-targetingcomplex at the site of a target RNA sequence. In the context offormation of an RNA-targeting complex, “target sequence” refers to anRNA sequence to which an RNA-targeting guide RNA is designed to havecomplementarity, where hybridization between an target sequence and anRNA-targeting guide RNA promotes the formation of an RNA-targetingcomplex. In some embodiments, a target sequence is located in thenucleus or cytoplasm of a cell.

In embodiments of the invention the terms guide sequence and guide RNAare used interchangeably as in foregoing cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art. A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome.

As used herein, the term “RNA-targeting guide RNA” is any polynucleotidesequence having sufficient complementarity with a target RNA sequence tohybridize with the target RNA sequence and direct sequence-specificbinding of an RNA-targeting complex to the target RNA sequence. In someembodiments, the degree of complementarity between an RNA-targetingguide sequence (within an RNA-targeting guide RNA) and its correspondingtarget RNA sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X,BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).The ability of an RNA-targeting guide sequence (within an RNA-targetingguide RNA) to direct sequence-specific binding of an RNA-targetingcomplex to a target RNA sequence may be assessed by any suitable assay.For example, the components of an RNA-targeting CRISPR system sufficientto form an RNA-targeting complex, including the RNA-targeting guidesequence to be tested, may be provided to a host cell having thecorresponding target RNA sequence, such as by transfection with vectorsencoding the components of the RNA-targeting complex, followed by anassessment of preferential targeting (e.g., cleavage) within the targetRNA sequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target RNA sequence may be evaluated in a test tube byproviding the target RNA sequence, components of an RNA-targetingcomplex, including the guide sequence to be tested and a control guidesequence different from the test guide sequence, and comparing bindingor rate of cleavage at the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art. An RNA-targeting guide sequence, and hence anRNA-targeting guide RNA may be selected to target any target RNAsequence. The target sequence is any RNA sequence. In some embodiments,the target sequence may be a sequence within an RNA molecule selectedfrom the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomaalRNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interferingRNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA),double stranded RNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA(IncRNA), and small cytoplasmatic RNA (scRNA). In some preferredembodiments, the target sequence may be a sequence within an RNAmolecule selected from the group consisting of mRNA, pre-mRNA, and rRNA.In some preferred embodiments, the target sequence may be a sequencewithin an RNA molecule selected from the group consisting of ncRNA, andIncRNA. In some more preferred embodiments, the target sequence may be asequence within an mRNA molecule or a pre-mRNA molecule.

In some embodiments, an RNA-targeting guide RNA is selected to reducethe degree secondary structure within the RNA-targeting guide RNA. Insome embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, 1%, or fewer of the nucleotides of the RNA-targeting guideRNA participate in self-complementary base pairing when optimallyfolded. Optimal folding may be determined by any suitable polynucleotidefolding algorithm. Some programs are based on calculating the minimalGibbs free energy. An example of one such algorithm is mFold, asdescribed by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148).Another example folding algorithm is the online webserver RNAfold,developed at Institute for Theoretical Chemistry at the University ofVienna, using the centroid structure prediction algorithm (see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church,2009, Nature Biotechnology 27(12): 1151-62).

In particular embodiments, the RNA-targeting guide RNA as taught hereincomprises a small RNA-targeting CRISPR/Cas system associated sequence(“sca”) and an RNA-targeting trans-activating CRISPR/Cas system sequence(“RNA-targeting tracr”).

The “scaRNA” sequence includes any polynucleotide sequence that hassufficient complementarity with an RNA-targeting tracrRNA sequence tohybridize. The “tracrRNA” sequence includes any polynucleotide sequencethat has sufficient complementarity with an RNA-targeting scaRNAsequence to hybridize. In general, degree of complementarity is withreference to the optimal alignment of the sca sequence and tracrsequence, along the length of the shorter of the two sequences. Optimalalignment may be determined by any suitable alignment algorithm, and mayfurther account for secondary structures, such as self-complementaritywithin either the sca sequence or tracr sequence. In some embodiments,the degree of complementarity between the tracr sequence and scasequence along the length of the shorter of the two when optimallyaligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 97.5%, 99%, or higher. To enable heterologous expression ofthe Cas9 protein and associated small RNAs (tracrRNA, scaRNA, and crRNA)from Francisella Novicida, Applicants introduce these elements into theE. coli genome via “clonetegration” (St-Pierre, F. et al. ACS Synth Biol2, 537-541 (2013).), a one step technique for cloning andrecombinase-mediated integration. Applicants also clonetegrate the DNAfor the endogenous RNA target of the FnCas9 protein in F. Novicida,FTN_1103. For verification of proper expression of both the Cas9 proteinand small RNA, Applicants detect the presence of RNA via qPCR andexpression of protein with western blot.

Applicants assay for activity of the FnCas9 on FTN_1103 in theheterologous system by measuring the difference in expression ofFTN_1103 with and without a functioning FnCas9 system. Applicants createa non-functioning system by omission of FnCas9, scaRNA, or tracrRNA. Bycomparing FTN_1103 expression, with qPCR, between functioning andnon-functioning heterologous systems, Applicants quantify the level ofknockdown due to FnCas9.

Applicants also perform a challenge experiment to verify the DNAtargeting and cleaving capability of FnCas9. This experiment closelyparallels similar work in E. coli for the heterologous expression ofStCas9 (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)).Applicants introduce a plasmid containing both a PAM and a resistancegene into the heterologous E. coli, and then plate on the correspondingantibiotic. If there is DNA cleavage of the plasmid, Applicants observeno viable colonies.

In some embodiments, the degree of complementarity between theRNA-targeting tracr sequence and RNA-targeting sca sequence along thelength of the shorter of the two when optimally aligned is about or morethan about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, orhigher. In some embodiments, the RNA-targeting tracr sequence is aboutor more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, 50, or more nucleotides in length. In someembodiments, the RNA-targeting tracr sequence and RNA-targeting scasequence are contained within a single transcript, such thathybridization between the two produces a transcript having a secondarystructure, such as a hairpin. In an embodiment of the invention, thetranscript or transcribed polynucleotide sequence has at least two ormore hairpins. In preferred embodiments, the transcript has two, three,four or five hairpins. In a further embodiment of the invention, thetranscript has at most five hairpins. In a hairpin structure the portionof the sequence 5′ of the final “N” and upstream of the loop correspondsto the tracr mate sequence, and the portion of the sequence 3′ of theloop corresponds to the tracr sequence Further non-limiting examples ofsingle polynucleotides comprising an RNA-targeting guide RNA, anRNA-targeting sca sequence, and an RNA-targeting tracr sequence are asfollows (listed 5′ to 3′), where “N” represents a base of a guidesequence, the first block of lower case letters represent the tracr matesequence, and the second block of lower case letters represent the tracrsequence, and the final poly-T sequence represents the transcriptionterminator: (1)NNNNNNNNNNNNNNttttgtacttcaagatttaGAAAtaaatttcagaagctacaaagatttttttttaaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ IDNO: 1); (2)NNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 2); (3)NNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 3); (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 4); (5)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 5); and (6)NNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT TTTTTT (SEQ IDNO: 6). Advantageously, the Cas9 isolated from Fracisella novicida(FnCas9) is contemplated for the present invention.

In some embodiments, candidate tracrRNA may be subsequently predicted bysequences that fulfill any or all of the following criteria: 1. sequencehomology to direct repeats (motif search in Geneious with up to 18-bpmismatches); 2. presence of a predicted Rho-independent transcriptionalterminator in direction of transcription; and 3. stable hairpinsecondary structure between tracrRNA and direct repeat. In someembodiments, 2 of these criteria may be used, for instance 1 and 2, 2and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In some embodiments, chimeric synthetic guide RNAs (sgRNAs) designs mayincorporate at least 12 bp of duplex structure between the RNA-targetingsca RNA and RNA-targeting tracrRNA.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of RNA-targeting guide RNA delivered.Optimal concentrations of RNA-targeting guide RNA can be determined bytesting different concentrations in a cellular or non-human eukaryoteanimal model and using deep sequencing the analyze the extent ofmodification at potential off-target genomic loci. The concentrationthat gives the highest level of on-target modification while minimizingthe level of off-target modification should be chosen for in vivodelivery. The RNA-targeting system is derived advantageously from a typeII CRISPR system. In some embodiments, one or more elements of anRNA-targeting system is derived from a particular organism comprising anendogenous RNA-targeting system, such as from Francisella novicida. Inpreferred embodiments of the invention, the RNA-targeting system is atype II CRISPR system and the Cas enzyme is Cas9. In particularembodiments, the Type II RNA-targeting Cas enzyme is a Type IIRNA-targeting Cas9 isolated from or derived from Francisella novocida.Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,homologues thereof, or modified versions thereof. In embodiments, theFrancisella novocida Cas9 (FnCas9) protein as referred to herein alsoencompasses a homologue or an orthologue of FnCas9. The terms“orthologue” (also referred to as “ortholog” herein) and “homologue”(also referred to as “homolog” herein) are well known in the art. Bymeans of further guidance, a “homologue” of a protein as used herein isa protein of the same species which performs the same or a similarfunction as the protein it is a homologue of. Homologs and orthologs maybe identified by homology modelling (see, e.g., Greer, Science vol. 228(1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or“structural BLAST” (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a“structural BLAST”: using structural relationships to infer function.Protein Sci. 2013 April; 22(4):359-66. doi: 10.1002/pro.2225.). See alsoShmakov et al. (2015) for application in the field of CRISPR-Cas loci.Homologous proteins may but need not be structurally related, or areonly partially structurally related. An “orthologue” of a protein asused herein is a protein of a different species which performs the sameor a similar function as the protein it is an orthologue of. Orthologousproteins may but need not be structurally related, or are only partiallystructurally related. In particular embodiments, the homologue ororthologue of FnCas9 as referred to herein has a sequence homology oridentity of at least 80%, more preferably at least 85%, even morepreferably at least 90%, such as for instance at least 95% with FnCas9.In further embodiments, the homologue or orthologue of FnCas9 asreferred to herein has a sequence identity of at least 80%, morepreferably at least 85%, even more preferably at least 90%, such as forinstance at least 95% with the wild type FnCas9. Where the FnCas9 hasone or more mutations (mutated), the homologue or orthologue of saidFnCas9 as referred to herein has a sequence identity of at least 80%,more preferably at least 85%, even more preferably at least 90%, such asfor instance at least 95% with the mutated FnCas9.

In an embodiment, the Type II RNA-targeting Cas protein may be a FnCas9ortholog of a genus which includes but is not limited to Corynebacter,Sutterella, Legionella, Treponema, Filifactor, Eubacterium,Streptococcus, Lactobacillus, ycoplasma, Bacteroides, Flaviivola,Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter,Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor,Mycoplasma and Campylobacter.

Methods of identifying orthologs of FnCas9 may involve identifying tracrsequences in genomes of interest. Identification of tracr sequences mayrelate to the following steps: Search for the direct repeats or tracrmate sequences in a database to identify a CRISPR region comprising aCRISPR enzyme. Search for homologous sequences in the CRISPR regionflanking the CRISPR enzyme in both the sense and antisense directions.Look for transcriptional terminators and secondary structures. Identifyany sequence that is not a direct repeat or a tracr mate sequence buthas more than 50% identity to the direct repeat or tracr mate sequenceas a potential tracr sequence. Take the potential tracr sequence andanalyze for transcriptional terminator sequences associated therewith.

In embodiments, the Francisella novocida Cas9 (FnCas9) protein asreferred to herein also encompasses a functional variant of FnCas9 or ahomologue or an orthologue thereof. A “functional variant” of a proteinas used herein refers to a variant of such protein which retains atleast partially the activity of that protein. Functional variants mayinclude mutants (which may be insertion, deletion, or replacementmutants), including polymorphs, etc. Also included within functionalvariants are fusion products of such protein with another, usuallyunrelated, nucleic acid, protein, polypeptide or peptide. Functionalvariants may be naturally occurring or may be man-made.

In an embodiment, the Type II RNA-targeting Cas protein, in particularFnCas9 or an ortholog or homolog thereof, may be codon-optimized forexpression in an eukaryotic cell. In an embodiment, the Type IIRNA-targeting Cas protein, in particular FnCas9 or an ortholog orhomolog thereof, may comprise one or more mutations, for example two ormore mutations, for example three or more mutations. The mutations maybe artificially introduced mutations and may include but are not limitedto one or more mutations in a catalytic domain. Examples of catalyticdomains with reference to a Cas9 enzyme may include but are not limitedto RuvC I, RuvC II, RuvC III and HNH domains.

In embodiments, the Francisella novocida Cas9 (FnCas9) protein asreferred to herein also encompasses a functional variant of FnCas9 or ahomologue or an orthologue thereof. A “functional variant” of a proteinas used herein refers to a variant of such protein which retains atleast partially the activity of that protein. Functional variants mayinclude mutants (which may be insertion, deletion, or replacementmutants), including polymorphs, etc. Also included within functionalvariants are fusion products of such protein with another, usuallyunrelated, nucleic acid, protein, polypeptide or peptide. Functionalvariants may be naturally occurring or may be man-made.

In an embodiment, the Type II RNA-targeting Cas protein, in particularFnCas9 or an ortholog or homolog thereof, may be codon-optimized forexpression in an eukaryotic cell. In an embodiment, the Type IIRNA-targeting Cas protein, in particular FnCas9 or an ortholog orhomolog thereof, may comprise one or more mutations, for example two ormore mutations, for example three or more mutations. The mutations maybe artificially introduced mutations and may include but are not limitedto one or more mutations in a catalytic domain. Examples of catalyticdomains with reference to a Cas9 enzyme may include but are not limitedto RuvC I, RuvC II, RuvC III and HNH domains.

In an embodiment, the Type II RNA-targeting Cas protein, in particularFnCas9 or an ortholog or homolog thereof, may be used as a generic RNAbinding protein with fusion to or being operably linked to a functionaldomain. Exemplary functional domains may include but are not limited totranslational initiator, translational activator, translationalrepressor, nucleases, in particular ribonucleases, a spliceosome, beads,a light inducible/controllable domain or a chemicallyinducible/controllable domain.

In some embodiments, the unmodified RNA-targeting Cas protein may havecleavage activity. In some embodiments, the RNA-targeting Cas proteinmay direct cleavage of one or both RNA strands at the location of atarget sequence, such as within the target sequence and/or within thecomplement of the target sequence. In some embodiments, theRNA-targeting Cas protein may direct cleavage of one or both RNA strandswithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200,500, or more base pairs from the first or last nucleotide of a targetsequence. In some embodiments, a vector encodes an RNA-targeting Casprotein that may be mutated with respect to a corresponding wild-typeenzyme such that the mutated RNA-targeting Cas protein lacks the abilityto cleave one or both RNA strands of a target polynucleotide containinga target sequence. As a further example, two or more catalytic domainsof Cas9 (RuvC I, RuvC II, and RuvC III or the HNH domain) may be mutatedto produce a mutated Cas9 substantially lacking all RNA cleavageactivity. In some embodiments, an RNA-targeting Cas protein may beconsidered to substantially lack all RNA cleavage activity when the RNAcleavage activity of the mutated enzyme is about no more than 25%, 10%,5%, 1%, 0.1%, 0.01%, or less of the RNA cleavage activity of thenon-mutated form of the enzyme; an example can be when the RNA cleavageactivity of the mutated form is nil or negligible as compared with thenon-mutated form. A Cas enzyme may be identified Cas9 as this can referto the general class of enzymes that share homology to the biggestnuclease with multiple nuclease domains from the type II CRISPR system.Most preferably, the Cas9 enzyme is from Francisella novicida (FnCas9 orFnCas9), or is derived from FnCas9. By derived. Applicants mean that thederived enzyme is largely based, in the sense of having a high degree ofsequence homology with, a wildtype enzyme, but that it has been mutated(modified) in some way as known in the art or as described herein. Itwill be appreciated that the terms Cas and CRISPR enzyme are generallyused herein interchangeably, unless otherwise apparent. As mentionedabove, many of the residue numberings used herein refer to the Cas9enzyme from the type II CRISPR locus in Francisella novicida. However,it will be appreciated that this invention includes many more Cas9s fromother species of microbes. In certain embodiments, Cas9 may beconstitutively present or inducibly present or conditionally present oradministered or delivered. Cas9 optimization may be used to enhancefunction or to develop new functions, one can generate chimeric Cas9proteins. And Cas9 may be used as a generic RNA binding protein.

Typically, in the context of an endogenous RNA-targeting system,formation of an RNA-targeting complex (comprising an RNA-targeting guideRNA hybridized to a target sequence and complexed with one or moreRNA-targeting Cas proteins) results in cleavage of one or both RNAstrands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the target sequence.

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed, see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding an RNA-targeting Cas protein is codonoptimized for expression in particular cells, such as eukaryotic cells.The eukaryotic cells may be those of or derived from a particularorganism, such as a mammal, including but not limited to human, ornon-human eukaryote or animal or mammal as herein discussed, e.g.,mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. Insome embodiments, processes for modifying the germ line genetic identityof human beings and/or processes for modifying the genetic identity ofanimals which are likely to cause them suffering without any substantialmedical benefit to man or animal, and also animals resulting from suchprocesses, may be excluded. In general, codon optimization refers to aprocess of modifying a nucleic acid sequence for enhanced expression inthe host cells of interest by replacing at least one codon (e.g., aboutor more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) ofthe native sequence with codons that are more frequently or mostfrequently used in the genes of that host cell while maintaining thenative amino acid sequence. Various species exhibit particular bias forcertain codons of a particular amino acid. Codon bias (differences incodon usage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” availableat www.kazusa.orjp/codon/ and these tables can be adapted in a number ofways. See Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, Pa.), are alsoavailable. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding anRNA-targeting Cas protein correspond to the most frequently used codonfor a particular amino acid.

In some embodiments, a vector encodes an RNA-targeting Cas proteincomprising one or more nuclear localization sequences (NLSs), such asabout or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. Insome embodiments, the RNA-targeting Cas protein comprises about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near theamino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more NLSs at or near the carboxy-terminus, or a combination of these(e.g., zero or at least one or more NLS at the amino-terminus and zeroor at one or more NLS at the carboxy terminus). When more than one NLSis present, each may be selected independently of the others, such thata single NLS may be present in more than one copy and/or in combinationwith one or more other NLSs present in one or more copies. In someembodiments, an NLS is considered near the N- or C-terminus when thenearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 40, 50, or more amino acids along the polypeptide chain from theN- or C-terminus. Non-limiting examples of NLSs include an NLS sequencederived from: the NLS of the SV40 virus large T-antigen, having theamino acid sequence PKKKRKV (SEQ ID NO: 7); the NLS from nucleoplasmin(e.g., the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK (SEQ ID NO: 8)); the c-myc NLS having the amino acidsequence PAAKRVKLD (SEQ ID NO: 9) or RQRRNELKRSP (SEQ ID NO: 10); thehRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 11); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV(SEQ ID NO: 12) of the IBB domain from importin-alpha; the sequencesVSRKRPRP (SEQ ID NO: 13) and PPKKARED (SEQ ID NO: 14) of the myoma Tprotein; the sequence PQPKKKPL (SEQ ID NO: 15) of human p53; thesequence SALIKKKKKMAP (SEQ ID NO: 16) of mouse c-abl IV; the sequencesDRLRR (SEQ ID NO: 17) and PKQKKRK (SEQ ID NO: 18) of the influenza virusNS1; the sequence RKLKKKIKKL (SEQ ID NO: 19) of the Hepatitis virusdelta antigen; the sequence REKKKFLKRR (SEQ ID NO: 20) of the mouse Mxprotein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 21) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 22) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the RNA-targeting Cas protein in a detectable amount inthe nucleus of a eukaryotic cell. In general, strength of nuclearlocalization activity may derive from the number of NLSs in theRNA-targeting Cas protein, the particular NLS(s) used, or a combinationof these factors. Detection of accumulation in the nucleus may beperformed by any suitable technique. For example, a detectable markermay be fused to the RNA-targeting protein, such that location within acell may be visualized, such as in combination with a means fordetecting the location of the nucleus (e.g., a stain specific for thenucleus such as DAPI). Cell nuclei may also be isolated from cells, thecontents of which may then be analyzed by any suitable process fordetecting protein, such as immunohistochemistry, Western blot, or enzymeactivity assay. Accumulation in the nucleus may also be determinedindirectly, such as by an assay for the effect of RNA-targeting complexformation (e.g., assay for RNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected byRNA-targeting complex formation and/or RNA-targeting Cas proteinactivity), as compared to a control not exposed to the RNA-targeting Casprotein or RNA-targeting complex, or exposed to an RNA-targeting Casprotein lacking the one or more NLSs.

In some embodiments, one or more vectors driving expression of one ormore elements of an RNA-targeting system are introduced into a host cellsuch that expression of the elements of the RNA-targeting system directformation of an RNA-targeting complex at one or more target sites. Forexample, an RNA-targeting Cas enzyme and an RNA-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. Or, RNA(s) of the RNA-targeting system can bedelivered to a transgenic RNA-targeting Cas9 animal or mammal, e.g., ananimal or mammal that constitutively or inducibly or conditionallyexpresses RNA-targeting Cas9; or an animal or mammal that is otherwiseexpressing RNA-targeting Cas9 or has cells containing RNA-targetingCas9, such as by way of prior administration thereto of a vector orvectors that code for and express in vivo RNA-targeting Cas9.Alternatively, two or more of the elements expressed from the same ordifferent regulatory elements, may be combined in a single vector, withone or more additional vectors providing any components of theRNA-targeting system not included in the first vector. RNA-targetingsystem elements that are combined in a single vector may be arranged inany suitable orientation, such as one element located 5′ with respect to(“upstream” of) or 3′ with respect to (“downstream” of) a secondelement. The coding sequence of one element may be located on the sameor opposite strand of the coding sequence of a second element, andoriented in the same or opposite direction. In some embodiments, asingle promoter drives expression of a transcript encoding anRNA-targeting Cas protein and the RNA-targeting guide RNA, embeddedwithin one or more intron sequences (e.g., each in a different intron,two or more in at least one intron, or all in a single intron). In someembodiments, the RNA-targeting Cas protein and the RNA-targeting guideRNA may be operably linked to and expressed from the same promoter.Delivery vehicles, vectors, particles, nanoparticles, formulations andcomponents thereof for expression of one or more elements of anRNA-targeting system are as used in the foregoing documents, such as WO2014/093622 (PCT/US2013/074667). In some embodiments, a vector comprisesone or more insertion sites, such as a restriction endonucleaserecognition sequence (also referred to as a “cloning site”). In someembodiments, one or more insertion sites (e.g., about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are locatedupstream and/or downstream of one or more sequence elements of one ormore vectors. In some embodiments, a vector comprises an insertion siteupstream of a tracr mate sequence, and optionally downstream of aregulatory element operably linked to the tracr mate sequence, such thatfollowing insertion of a guide sequence into the insertion site and uponexpression the guide sequence directs sequence-specific binding of anRNA-targeting complex to a target sequence in a eukaryotic cell. In someembodiments, a vector comprises two or more insertion sites, so as toallow insertion of a guide sequence at each site. In such anarrangement, the two or more guide sequences may comprise two or morecopies of a single guide sequence, two or more different guidesequences, or combinations of these. When multiple different guidesequences are used, a single expression construct may be used to targetRNA-targeting activity to multiple different, corresponding targetsequences within a cell. For example, a single vector may comprise aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guidesequences. In some embodiments, about or more than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may beprovided, and optionally delivered to a cell. In some embodiments, avector comprises a regulatory element operably linked to anenzyme-coding sequence encoding an RNA-targeting Cas protein.RNA-targeting Cas protein or RNA-targeting guide RNA or RNA(s) can bedelivered separately; and advantageously at least one of these isdelivered via a nanoparticle complex. RNA-targeting Cas protein mRNA canbe delivered prior to the RNA-targeting guide RNA to give time forRNA-targeting Cas protein to be expressed. RNA-targeting Cas proteinmRNA might be administered 1-12 hours (preferably around 2-6 hours)prior to the administration of RNA-targeting guide RNA. Alternatively,RNA-targeting Cas protein mRNA and RNA-targeting guide RNA can beadministered together. Advantageously, a second booster dose ofRNA-targeting guide RNA can be administered 1-12 hours (preferablyaround 2-6 hours) after the initial administration of RNA-targeting Casprotein mRNA+RNA-targeting guide RNA. Additional administrations ofRNA-targeting Cas protein mRNA and/or RNA-targeting guide RNA might beuseful to achieve the most efficient levels of genome modification.

In one aspect, the invention provides methods for using one or moreelements of an RNA-targeting system. The RNA-targeting complex of theinvention provides an effective means for modifying a target RNA. TheRNA-targeting complex of the invention has a wide variety of utilityincluding modifying (e.g., deleting, inserting, translocating,inactivating, activating) a target RNA in a multiplicity of cell types.As such the RNA-targeting complex of the invention has a broad spectrumof applications in, e.g., gene therapy, drug screening, diseasediagnosis, and prognosis. An exemplary RNA-targeting complex comprisesan RNA-targeting Cas protein complexed with an RNA-targeting guide RNAhybridized to a target sequence within the target RNA. In oneembodiment, this invention provides a method of cleaving a target RNA.The method may comprise modifying a target RNA using an RNA-targetingcomplex that binds to the target RNA and effect cleavage of said targetRNA. In an embodiment, the RNA-targeting complex of the invention, whenintroduced into a cell, may create a break (e.g., a single or a doublestrand break) in the RNA sequence. For example, the method can be usedto cleave a disease RNA in a cell For example, an exogenous RNA templatecomprising a sequence to be integrated flanked by an upstream sequenceand a downstream sequence may be introduced into a cell. The upstreamand downstream sequences share sequence similarity with either side ofthe site of integration in the RNA. Where desired, a donor RNA can bemRNA. The exogenous RNA template comprises a sequence to be integrated(e.g., a mutated RNA). The sequence for integration may be a sequenceendogenous or exogenous to the cell. Examples of a sequence to beintegrated include RNA encoding a protein or a non-coding RNA (e.g., amicroRNA). Thus, the sequence for integration may be operably linked toan appropriate control sequence or sequences. Alternatively, thesequence to be integrated may provide a regulatory function. Theupstream and downstream sequences in the exogenous RNA template areselected to promote recombination between the RNA sequence of interestand the donor RNA. The upstream sequence is an RNA sequence that sharessequence similarity with the RNA sequence upstream of the targeted sitefor integration. Similarly, the downstream sequence is an RNA sequencethat shares sequence similarity with the RNA sequence downstream of thetargeted site of integration. The upstream and downstream sequences inthe exogenous RNA template can have 75%, 80%, 85%, 90%, 95%, or 100%sequence identity with the targeted RNA sequence. Preferably, theupstream and downstream sequences in the exogenous RNA template haveabout 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with thetargeted RNA sequence. In some methods, the upstream and downstreamsequences in the exogenous RNA template have about 99% or 100% sequenceidentity with the targeted RNA sequence. An upstream or downstreamsequence may comprise from about 20 bp to about 2500 bp, for example,about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,or 2500 bp. In some methods, the exemplary upstream or downstreamsequence have about 200 bp to about 2000 bp, about 600 bp to about 1000bp, or more particularly about 700 bp to about 1000 bp. In some methods,the exogenous RNA template may further comprise a marker. Such a markermay make it easy to screen for targeted integrations. Examples ofsuitable markers include restriction sites, fluorescent proteins, orselectable markers. The exogenous RNA template of the invention can beconstructed using recombinant techniques (see, for example, Sambrook etal., 2001 and Ausubel et al., 1996). In a method for modifying a targetRNA by integrating an exogenous RNA template, a break (e.g., doublestranded break in dsRNA) is introduced into the RNA sequence by theRNA-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of an RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using an RNA-targeting complexthat binds to the RNA (e.g., mRNA or pre-mRNA). In some methods, atarget RNA can be inactivated to effect the modification of theexpression in a cell. For example, upon the binding of an RNA-targetingcomplex to a target sequence in a cell, the target RNA is inactivatedsuch that the sequence is not translated, the coded protein is notproduced, or the sequence does not function as the wild-type sequencedoes. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of an RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be an RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, IncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be an RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe an RNA transcribed from a gene that becomes expressed at anabnormally low level, where the altered expression correlates with theoccurrence and/or progression of the disease. A disease-associated RNAalso refers to an RNA transcribed from a gene possessing mutation(s) orgenetic variation that is directly responsible or is in linkagedisequilibrium with a gene(s) that is responsible for the etiology of adisease. The translated products may be known or unknown, and may be ata normal or abnormal level. The target RNA of an RNA-targeting complexcan be any RNA endogenous or exogenous to the eukaryotic cell. Forexample, the target RNA can be an RNA residing in the nucleus of theeukaryotic cell. The target RNA can be a sequence (e.g., mRNA orpre-mRNA) coding a gene product (e.g., a protein) or a non-codingsequence (e.g., ncRNA, IncRNA, tRNA, or rRNA).

In some embodiments, the method may comprise allowing an RNA-targetingcomplex to bind to the target RNA to effect cleavage of said target RNAthereby modifying the target RNA, wherein the RNA-targeting complexcomprises an RNA-targeting Cas protein complexed with an RNA-targetingguide RNA hybridized to a target sequence within said target RNA. In oneaspect, the invention provides a method of modifying expression of anRNA in a eukaryotic cell. In some embodiments, the method comprisesallowing an RNA-targeting complex to bind to the RNA such that saidbinding results in increased or decreased expression of said RNA;wherein the RNA-targeting complex comprises an RNA-targeting Cas proteincomplexed with an RNA-targeting guide RNA. Similar considerations andconditions apply as above for methods of modifying a target RNA. Infact, these sampling, culturing and re-introduction options apply acrossthe aspects of the present invention. In one aspect, the inventionprovides for methods of modifying a target RNA in a eukaryotic cell,which may be in vivo, ex vivo or in vitro. In some embodiments, themethod comprises sampling a cell or population of cells from a human ornon-human animal, and modifying the cell or cells. Culturing may occurat any stage ex vivo. The cell or cells may even be re-introduced intothe non-human animal or plant. For re-introduced cells it isparticularly preferred that the cells are stem cells.

Indeed, in any aspect of the invention, the RNA-targeting complex maycomprise an RNA-targeting Cas protein complexed with an RNA-targetingguide RNA hybridized to a target sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving RNA sequence targeting, that relate to the RNA-targetingsystem and components thereof. In advantageous embodiments, the Casenzyme is Cas9. In preferred embodiments, the Cas enzyme is FnCas9. Anadvantage of the present methods is that the CRISPR system minimizes oravoids off-target binding and its resulting side effects. This isachieved using systems arranged to have a high degree of sequencespecificity for the target RNA.

In relation to an RNA-targeting complex or system preferably, theRNA-targeting tracr sequence has one or more hairpins and is 30 or morenucleotides in length, 40 or more nucleotides in length, or 50 or morenucleotides in length; the RNA-targeting sca sequence is between 10 to30 nucleotides in length, the RNA-targeting Cas protein is a Type IICas9 enzyme.

The use of two different aptamers (distinct RNA-targeting guide RNAs)allows an activator-adaptor protein fusion and a repressor-adaptorprotein fusion to be used, with different RNA-targeting guide RNAs, toactivate expression of one RNA, whilst repressing another. They, alongwith their different RNA-targeting guide RNAs can be administeredtogether, or substantially together, in a multiplexed approach. A largenumber of such modified RNA-targeting guide RNAs can be used all at thesame time, for example 10 or 20 or 30 and so forth, whilst only one (orat least a minimal number) of Cas9s to be delivered, as a comparativelysmall number of Cas9s can be used with a large number modified guides.The adaptor protein may be associated (preferably linked or fused to)one or more activators or one or more repressors. For example, theadaptor protein may be associated with a first activator and a secondactivator. The first and second activators may be the same, but they arepreferably different activators. Three or more or even four or moreactivators (or repressors) may be used, but package size may limit thenumber being higher than 5 different functional domains. Linkers arepreferably used, over a direct fusion to the adaptor protein, where twoor more functional domains are associated with the adaptor protein.Suitable linkers might include the GlySer linker.

It is also envisaged that the RNA-targeting Cas protein-guide RNAcomplex as a whole may be associated with two or more functionaldomains. For example, there may be two or more functional domainsassociated with the RNA-targeting Cas protein, or there may be two ormore functional domains associated with the RNA-targeting guide RNA (viaone or more adaptor proteins), or there may be one or more functionaldomains associated with the RNA-targeting Cas protein and one or morefunctional domains associated with the RNA-targeting guide RNA (via oneor more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, GlySer linkers GGGS (SEQ ID NO: 23)can be used. They can be used in repeats of 3 ((GGGGS)₃ (SEQ ID NO: 24))or 6 (SEQ ID NO: 25), 9 (SEQ ID NO: 26) or even 12 (SEQ ID NO: 27) ormore, to provide suitable lengths, as required. Linkers can be usedbetween the RNA-targeting guide RNAs and the functional domain(activator or repressor), or between the RNA-targeting Cas protein(Cas9) and the functional domain (activator or repressor). The linkersthe user to engineer appropriate amounts of“mechanical flexibility”.

The invention comprehends an RNA-targeting complex comprising anRNA-targeting Cas protein and an RNA-targeting guide RNA, wherein theRNA-targeting Cas protein comprises at least one mutation, such that theRNA-targeting Cas protein has no more than 5% of the activity of theRNA-targeting Cas protein not having the at least one mutation and,optional, at least one or more nuclear localization sequences; theRNA-targeting guide RNA comprises a guide sequence capable ofhybridizing to a target sequence in an RNA of interest in a cell; andwherein: the RNA-targeting Cas protein is associated with two or morefunctional domains; or at least one loop of the RNA-targeting guide RNAis modified by the insertion of distinct RNA sequence(s) that bind toone or more adaptor proteins, and wherein the adaptor protein isassociated with two or more functional domains; or the RNA-targeting Casprotein is associated with one or more functional domains and at leastone loop of the RNA-targeting guide RNA is modified by the insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins, andwherein the adaptor protein is associated with one or more functionaldomains.

Delivery Generally

Vector delivery, e.g., plasmid, viral delivery: The RNA-targeting guideRNA, for instance a FnCas9, and/or an RNA-targeting guide RNA, can bedelivered using any suitable vector, e.g., plasmid or viral vectors,such as adeno associated virus (AAV), lentivirus, adenovirus or otherviral vector types, or combinations thereof. RNA-targeting Cas9 and oneor more RNA-targeting guide RNAs can be packaged into one or morevectors, e.g., plasmid or viral vectors. In some embodiments, thevector, e.g., plasmid or viral vector is delivered to the tissue ofinterest by, for example, an intramuscular injection, while other timesthe delivery is via intravenous, transdermal, intranasal, oral, mucosal,or other delivery methods. Such delivery may be either via a singledose, or multiple doses. One skilled in the art understands that theactual dosage to be delivered herein may vary greatly depending upon avariety of factors, such as the vector choice, the target cell,organism, or tissue, the general condition of the subject to be treated,the degree of transformation/modification sought, the administrationroute, the administration mode, the type of transformation/modificationsought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10⁰ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁸ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding anRNA-targeting Cas protein, operably linked to said promoter; (iii) aselectable marker; (iv) an origin of replication; and (v) atranscription terminator downstream of and operably linked to (ii). Theplasmid can also encode the RNA-targeting guide RNA, but one or more ofthese may instead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA-targeting guide RNAs are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver RNA-targeting Cas protein and RNA-targeting guideRNA into cells using liposomes or nanoparticles. Thus delivery of theRNA-targeting Cas protein, such as a Cas9 and/or delivery of theRNA-targeting guide RNAs of the invention may be in RNA form and viamicrovesicles, liposomes or nanoparticles. For example, Cas9 mRNA andRNA-targeting guide RNA can be packaged into liposomal particles fordelivery in vivo. Liposomal transfection reagents such as lipofectaminefrom Life Technologies and other reagents on the market can effectivelydeliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe RNA-targeting system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December, 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15.) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurify and characterized from transfected cell supernatant, then RNA isloaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (α-tocopherol) may be conjugated withRNA-targeting Cas protein and delivered to the brain along with highdensity lipoprotein (HDL), for example in a similar manner as was doneby Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for deliveringshort-interfering RNA (siRNA) to the brain. Mice were infused viaOsmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled withphosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL andconnected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannulawas placed about 0.5 mm posterior to the bregma at midline for infusioninto the dorsal third ventricle. Uno et al. found that as little as 3nmol of Toc-siRNA with HDL could induce a target reduction in comparabledegree by the same ICV infusion method. A similar dosage ofRNA-targeting Cas protein conjugated to α-tocopherol and co-administeredwith HDL targeted to the brain may be contemplated for humans in thepresent invention, for example, about 3 nmol to about 3 μmol ofRNA-targeting Cas protein targeted to the brain may be contemplated. Zouet al. ((HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a methodof lentiviral-mediated delivery of short-hairpin RNAs targeting PKCγ forin vivo gene silencing in the spinal cord of rats. Zou et al.administered about 10 μl of a recombinant lentivirus having a titer of1×10⁹ transducing units (TU)/ml by an intrathecal catheter. A similardosage of RNA-targeting Cas protein expressed in a lentiviral vectortargeted to the brain may be contemplated for humans in the presentinvention, for example, about 10-50 ml of RNA-targeting Cas proteintargeted to the brain in a lentivirus having a titer of 1×10⁹transducing units (TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g., byinjection. Injection can be performed stereotactically via a craniotomy.

Packaging and Promoters Generally

Ways to package RNA-targeting Cas protein (such as FnCas9) codingnucleic acid molecules, e.g., DNA, into vectors, e.g., viral vectors, tomediate genome modification in vivo include:

Single Virus Vector:

Vector containing two or more expression cassettes:

Promoter-RNA-targeting Cas protein coding nucleic acidmolecule-terminator

Promoter-RNA-targeting guide RNA1-terminator

Promoter-RNA-targeting guide RNA (N)-terminator (up to size limit ofvector)

Double Virus Vector:

Vector 1 containing one expression cassette for driving the expressionof RNA-targeting Cas protein (such as FnCas9)

Promoter-RNA-targeting Cas protein coding nucleic acidmolecule-terminator

Vector 2 containing one more expression cassettes for driving theexpression of one or more RNA-targeting guide RNAs

Promoter-RNA-targeting guide RNA1-terminator

Promoter-RNA-targeting guide RNA1 (N)-terminator (up to size limit ofvector)

To mediate homology-directed repair.

-   -   In addition to the single and double virus vector approaches        described above, an additional vector is used to deliver a        homology-direct repair template.

The promoter used to drive RNA-targeting Cas protein (such as FnCas9)coding nucleic acid molecule expression can include:

AAV ITR can serve as a promoter: this is advantageous for eliminatingthe need for an additional promoter element (which can take up space inthe vector). The additional space freed up can be used to drive theexpression of additional elements (gRNA, etc.). Also, ITR activity isrelatively weaker, so can be used to reduce potential toxicity due toover expression of RNA-targeting Cas protein (such as FnCas9).

For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40,Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: SynapsinI for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can use OG-2.

The promoter used to drive RNA-targeting guide RNA can include:

Pol III promoters such as U6 or H1

Use of Pol II promoter and intronic cassettes to express RNA-targetingguide RNA

Adeno Associated Virus (AAV)

RNA-targeting Cas protein (such as FnCas9) and one or more RNA-targetingguide RNA can be delivered using adeno associated virus (AAV),lentivirus, adenovirus or other plasmid or viral vector types, inparticular, using formulations and doses from, for example, U.S. Pat.No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat. No.8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946(formulations, doses for DNA plasmids) and from clinical trials andpublications regarding the clinical trials involving lentivirus, AAV andadenovirus. For examples, for AAV, the route of administration,formulation and dose can be as in U.S. Pat. No. 8,454,972 and as inclinical trials involving AAV. For Adenovirus, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g., a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of RNA-targeting Cas protein (such asFnCas9) can be driven by a cell-type specific promoter. For example,liver-specific expression might use the Albumin promoter andneuron-specific expression (e.g., for targeting CNS disorders) might usethe Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response) and    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means thatRNA-targeting Cas protein (such as FnCas9) as well as a promoter andtranscription terminator have to be all fit into the same viral vector.Therefore embodiments of the invention include utilizing homologs ofRNA-targeting Cas protein (such as FnCas9) that are shorter.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.Sug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4 C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinffctious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the RNA-targeting system of the presentinvention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the RNA-targeting system of the presentinvention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patient weightmay be collected and prestimulated for 16 to 20 hours in X-VIVO 15medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The RNA-targeting Cas protein, for instance a FnCas9,and/or RNA-targeting guide RNA, can also be delivered in the form ofRNA. RNA-targeting Cas protein (such as FnCas9) mRNA can be generatedusing in vitro transcription. For example, RNA-targeting Cas protein(such as FnCas9) mRNA can be synthesized using a PCR cassette containingthe following elements: T7_promoter-kozak sequence (GCCACC)-Cas9-3′ UTRfrom beta globin-polyA tail (a string of 120 or more adenines). Thecassette can be used for transcription by T7 polymerase. RNA-targetingguide RNAs can also be transcribed using in vitro transcription from acassette containing T7_promoter-GG-RNA-targeting guide RNA sequence.

To enhance expression and reduce possible toxicity, the RNA-targetingCas protein-coding sequence and/or the RNA-targeting guide RNA can bemodified to include one or more modified nucleoside e.g., using pseudo-Uor 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Nanoparticles

RNA-targeting Cas protein (such as FnCas9) mRNA and RNA-targeting guideRNA may be delivered simultaneously using nanoparticles or lipidenvelopes.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured nanoparticles with a poly(β-amino ester) (PBAE) coreenveloped by a phospholipid bilayer shell. These were developed for invivo mRNA delivery. The pH-responsive PBAE component was chosen topromote endosome disruption, while the lipid surface layer was selectedto minimize toxicity of the polycation core. Such are, therefore,preferred for delivering RNA of the present invention.

In one embodiment, nanoparticles based on self assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

In one embodiment, nanoparticles that can deliver RNA to a cancer cellto stop tumor growth developed by Dan Anderson's lab at MIT may beused/and or adapted to the RNA-targeting system of the presentinvention. In particular, the Anderson lab developed fully automated,combinatorial systems for the synthesis, purification, characterization,and formulation of new biomaterials and nanoformulations. See, e.g.,Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6;Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the RNA-targeting system of the present invention.In one aspect, the aminoalcohol lipidoid compounds are combined with anagent to be delivered to a cell or a subject to form microparticles,nanoparticles, liposomes, or micelles. The agent to be delivered by theparticles, liposomes, or micelles may be in the form of a gas, liquid,or solid, and the agent may be a polynucleotide, protein, peptide, orsmall molecule. The minoalcohol lipidoid compounds may be combined withother aminoalcohol lipidoid compounds, polymers (synthetic or natural),surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to formthe particles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the RNA-targetingsystem of the present invention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe RNA-targeting system of the present invention. Doses of about 0.01to about 1 mg per kg of body weight administered intravenously arecontemplated. Medications to reduce the risk of infusion-relatedreactions are contemplated, such as dexamethasone, acetampinophen,diphenhydramine or cetirizine, and ranitidine are contemplated. Multipledoses of about 0.3 mg per kilogram every 4 weeks for five doses are alsocontemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding RNA-targeting Cas protein to the liver. A dosage of aboutfour doses of 6 mg/kg of the LNP every two weeks may be contemplated.Tabernero et al. demonstrated that tumor regression was observed afterthe first 2 cycles of LNPs dosed at 0.7 mg/kg, and by the end of 6cycles the patient had achieved a partial response with completeregression of the lymph node metastasis and substantial shrinkage of theliver tumors. A complete response was obtained after 40 doses in thispatient, who has remained in remission and completed treatment afterreceiving doses over 26 months. Two patients with RCC and extrahepaticsites of disease including kidney, lung, and lymph nodes that wereprogressing following prior therapy with VEGF pathway inhibitors hadstable disease at all sites for approximately 8 to 12 months, and apatient with PNET and liver metastases continued on the extension studyfor 18 months (36 doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orRNA-targeting RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and encapsulation may be used/and or adapted fromRosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200,December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificRNA-targeting complex RNA may be encapsulated in LNPs containingDLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationiclipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may beincorporated to assess cellular uptake, intracellular delivery, andbiodistribution. Encapsulation may be performed by dissolving lipidmixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG(40:10:40:10 molar ratio) in ethanol to a final lipid concentration of10 mmol/l. This ethanol solution of lipid may be added drop-wise to 50mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a finalconcentration of 30% ethanol vol/vol. Large unilamellar vesicles may beformed following extrusion of multilamellar vesicles through two stacked80 nm Nuclepore polycarbonate filters using the Extruder (NorthernLipids, Vancouver, Canada). Encapsulation may be achieved by adding RNAdissolved at 2 mg/ml in 50 mmol/1 citrate, pH 4.0 containing 30% ethanolvol/vol drop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Nanoparticle size distributionmay be determined by dynamic light scattering using a NICOMP 370particle sizer, the vesicle/intensity modes, and Gaussian fitting(Nicomp Particle Sizing, Santa Barbara, Calif.). The particle size forall three LNP systems may be ˜70 nm in diameter. RNA encapsulationefficiency may be determined by removal of free RNA using VivaPureDMiniH columns (Sartorius Stedim Biotech) from samples collected beforeand after dialysis. The encapsulated RNA may be extracted from theeluted nanoparticles and quantified at 260 nm. RNA to lipid ratio wasdetermined by measurement of cholesterol content in vesicles using theCholesterol E enzymatic assay from Wako Chemicals USA (Richmond, Va.).In conjunction with the herein discussion of LNPs and PEG lipids,PEGylated liposomes or LNPs are likewise suitable for delivery of anRNA-targeting system or components thereof.

Preparation of large LNPs may be used/and or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. Alipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at an RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-1 m syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other nanoparticles(particularly gold nanoparticles) are also contemplated as a means todelivery RNA-targeting system to intended targets. Significant data showthat AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs,based upon nucleic acid-functionalized gold nanoparticles, are useful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling nanoparticles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of RNA-targeting complex RNA is envisionedfor delivery in the self-assembling nanoparticles of Schiffelers et al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHS ester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNA sense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA nanoparticles maybe formed by using cyclodextrin-containing polycations. Typically,nanoparticles were formed in water at a charge ratio of 3 (+/−) and ansiRNA concentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted nanoparticles were modifiedwith Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5%(wt/vol) glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts an RNA clinicaltrial that uses a targeted nanoparticle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetednanoparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The nanoparticles comprise, consist essentiallyof, or consist of a synthetic delivery system containing: (1) a linear,cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF)targeting ligand displayed on the exterior of the nanoparticle to engageTF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilicpolymer (polyethylene glycol (PEG) used to promote nanoparticlestability in biological fluids), and (4) siRNA designed to reduce theexpression of the RRM2 (sequence used in the clinic was previouslydenoted siR2B+5). The TFR has long been known to be upregulated inmalignant cells, and RRM2 is an established anticancer target. Thesenanoparticles (clinical version denoted as CALAA-01) have been shown tobe well tolerated in multi-dosing studies in non-human primates.Although a single patient with chronic myeloid leukaemia has beenadministered siRNA by liposomal delivery, Davis et al.'s clinical trialis the initial human trial to systemically deliver siRNA with a targeteddelivery system and to treat patients with solid cancer. To ascertainwhether the targeted delivery system can provide effective delivery offunctional siRNA to human tumours, Davis et al. investigated biopsiesfrom three patients from three different dosing cohorts; patients A, Band C, all of whom had metastatic melanoma and received CALAA-01 dosesof 18, 24 and 30 mg m⁻² siRNA, respectively. Similar doses may also becontemplated for the RNA-targeting system of the present invention. Thedelivery of the invention may be achieved with nanoparticles containinga linear, cyclodextrin-based polymer (CDP), a human transferrin protein(TF) targeting ligand displayed on the exterior of the nanoparticle toengage TF receptors (TFR) on the surface of the cancer cells and/or ahydrophilic polymer (for example, polyethylene glycol (PEG) used topromote nanoparticle stability in biological fluids).

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.,diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of RNA-targetingsystem e.g., RNA-targeting Cas protein or mRNA, or RNA-targeting guideRNA, or any combination thereof, and may include additional carriersand/or excipients) to provide particles of an optimal size for deliveryfor any in vitro, ex vivo and/or in vivo application of the presentinvention. In certain preferred embodiments, particle dimension (e.g.,diameter) characterization is based on measurements using dynamic laserscattering (DLS). Mention is made of U.S. Pat. No. 8,709,843; U.S. Pat.No. 6,007,845; U.S. Pat. No. 5,855,913; U.S. Pat. No. 5,985,309; U.S.Pat. No. 5,543,158; and the publication by James E. Dahlman and CarmenBarnes et al. Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84, concerning particles, methods of making andusing them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Nanoparticles

In terms of this invention, it is preferred to have one or morecomponents of RNA-targeting complex, e.g., RNA-targeting Cas protein ormRNA, or RNA-targeting guide RNA delivered using nanoparticles or lipidenvelopes. Other delivery systems or vectors are may be used inconjunction with the nanoparticle aspects of the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, nanoparticles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Nanoparticles encompassed in the present invention may be provided indifferent forms, e.g., as solid nanoparticles (e.g., metal such assilver, gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of nanoparticles, or combinations thereof. Metal,dielectric, and semiconductor nanoparticles may be prepared, as well ashybrid structures (e.g., core-shell nanoparticles). Nanoparticles madeof semiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft nanoparticles have been manufactured, and are withinthe scope of the present invention. A prototype nanoparticle ofsemi-solid nature is the liposome. Various types of liposomenanoparticles are currently used clinically as delivery systems foranticancer drugs and vaccines. Nanoparticles with one half hydrophilicand the other half hydrophobic are termed Janus particles and areparticularly effective for stabilizing emulsions. They can self-assembleat water/oil interfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid.

U.S. Pat. No. 6,007,845, incorporated herein by reference, providesparticles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material.

U.S. Pat. No. 5,855,913, incorporated herein by reference, provides aparticulate composition having aerodynamically light particles having atap density of less than 0.4 g/cm3 with a mean diameter of between 5 □μmand 30 μm, incorporating a surfactant on the surface thereof for drugdelivery to the pulmonary system.

U.S. Pat. No. 5,985,309, incorporated herein by reference, providesparticles incorporating a surfactant and/or a hydrophilic or hydrophobiccomplex of a positively or negatively charged therapeutic or diagnosticagent and a charged molecule of opposite charge for delivery to thepulmonary system.

U.S. Pat. No. 5,543,158, incorporated herein by reference, providesbiodegradable injectable nanoparticles having a biodegradable solid corecontaining a biologically active material and poly(alkylene glycol)moieties on the surface.

WO2012135025 (also published as US20120251560), incorporated herein byreference, describes conjugated polyethyleneimine (PEI) polymers andconjugated aza-macrocycles (collectively referred to as “conjugatedlipomer” or “lipomers”). In certain embodiments, it can envisioned thatsuch conjugated lipomers can be used in the context of the RNA-targetingsystem to achieve in vitro, ex vivo and in vivo genomic perturbations tomodify gene expression, including modulation of protein expression.

In one embodiment, the nanoparticle may be epoxide-modifiedlipid-polymer, advantageously 7C1 (see, e.g., James E. Dahlman andCarmen Barnes et al. Nature Nanotechnology (2014) published online 11May 2014, doi:10.1038/nnano.2014.84). C71 was synthesized by reactingC15 epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce nanoparticles (diameter between 35and 60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver theRNA-targeting system of the present invention to pulmonary,cardiovascular or renal cells, however, one of skill in the art mayadapt the system to deliver to other target organs. Dosage ranging fromabout 0.05 to about 0.6 mg/kg are envisioned. Dosages over several daysor weeks are also envisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by nanoparticle tracking analysis (NTA)and electron microscopy. Alvarez-Erviti et al. obtained 6-12 μg ofexosomes (measured based on protein concentration) per 10⁶ cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the RNA-targeting system of the present invention to therapeutictargets, especially neurodegenerative diseases. A dosage of about 100 to1000 mg of RNA-targeting system encapsulated in about 100 to 1000 mg ofRVG exosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7,2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of RNA-targeting system into exosomesmay be conducted similarly to siRNA. The exosomes may be co-culturedwith monocytes and lymphocytes isolated from the peripheral blood ofhealthy donors. Therefore, it may be contemplated that exosomescontaining RNA-targeting system may be introduced to monocytes andlymphocytes of and autologously reintroduced into a human. Accordingly,delivery or administration according to the invention may be performedusing plasma exosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro., Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi: 10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at accessible atcshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long. These particlesallow delivery of a transgene to the entire brain after an intravascularinjection. Without being bound by limitation, it is believed thatneutral lipid particles with specific antibodies conjugated to surfaceallow crossing of the blood brain barrier via endocytosis. Applicantpostulates utilizing Trojan Horse Liposomes to deliver the RNA-targetingsystem or complex to the brain via an intravascular injection, whichwould allow whole brain transgenic animals without the need forembryonic manipulation. About 1-5 g of DNA or RNA may be contemplatedfor in vivo administration in liposomes.

In another embodiment, the RNA-targeting system may be administered inliposomes, such as a stable nucleic-acid-lipid particle (SNALP) (see,e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August2005). Daily intravenous injections of about 1, 3 or 5 mg/kg/day of aspecific RNA-targeting system targeted in a SNALP are contemplated. Thedaily treatment may be over about three days and then weekly for aboutfive weeks. In another embodiment, a specific RNA-targeting systemencapsulated SNALP) administered by intravenous injection to at doses ofabout 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al.,Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may containthe lipids 3-N-[(wmethoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010, 375: 1896-905). A dosage of about 2 mg/kgtotal RNA-targeting systemper dose administered as, for example, a bolusintravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC)-both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≧0.4 mg/kg,all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of −5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the RNA-targeting system of the presentinvention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate RNA-targeting system or components thereof ornucleic acid molecule(s) coding therefor e.g., similar to SiRNA (see,e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and hencemay be employed in the practice of the invention. A preformed vesiclewith the following lipid composition may be contemplated: amino lipid,distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11±0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the RNA-targeting guide RNA. Particles containing thehighly potent amino lipid 16 may be used, in which the molar ratio ofthe four lipid components 16, DSPC, cholesterol and PEG-lipid(50/10/38.5/1.5) which may be further optimized to enhance in vivoactivity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume: 29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the RNA-targetingsystem of the present invention or component(s) thereof or nucleic acidmolecule(s) coding therefor to form lipid nanoparticles (LNPs). Lipidsinclude, but are not limited to, DLin-KC2-DMA4, C12-200 and colipidsdisteroylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated with RNA-targeting system instead of siRNA (see, e.g.,Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipidnanoparticles (LNPs), respectively. The formulations may have meanparticle diameters of ˜80 nm with >90% entrapment efficiency. A 3 mg/kgdose may be contemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The RNA-targeting system or components thereof or nucleic acidmolecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesized from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of RNA-targeting system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of RNA-targeting system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines.

(1) One day before treatment, plate 1×10⁵ cells per well in a 48-wellplate.

(2) On the day of treatment, dilute purified +36 GFP protein in serumfree media to a final concentration 200 nM. Add RNA to a finalconcentration of 50 nM. Vortex to mix and incubate at room temperaturefor 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of +36 GFP and RNA, add the protein-RNAcomplexes to cells.

(5) Incubate cells with complexes at 37° C. for 4 h.

(6) Following incubation, aspirate the media and wash three times with20 U/mL heparin PBS. Incubate cells with serum-containing media for afurther 48 h or longer depending upon the assay for activity.

(7) Analyze cells by immunoblot, qPCR, phenotypic assay, or otherappropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications.

(1) One day before treatment, plate 1×10⁵ per well in a 48-well plate.

(2) On the day of treatment, dilute purified 136 GFP protein in serumfree media to a final concentration 2 mM. Add 1 mg of plasmid DNA.Vortex to mix and incubate at room temperature for 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of

36 GFP and plasmid DNA, gently add the protein-DNA complexes to cells.

(5) Incubate cells with complexes at 37 C for 4 h.

(6) Following incubation, aspirate the media and wash with PBS. Incubatecells in serum-containing media and incubate for a further 24-48 h.

(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression)as appropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe RNA-targeting system of the present invention. These systems of Dr.Lui and documents herein in conjunction with herein teachings can beemployed in the delivery of RNA-targeting system(s) or component(s)thereof or nucleic acid molecule(s) coding therefor.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the RNA-targeting system or component(s) thereof or nucleicacid molecule(s) coding therefor. For example, US Patent Publication20110195123 discloses an implantable medical device which elutes a druglocally and in prolonged period is provided, including several types ofsuch a device, the treatment modes of implementation and methods ofimplantation. The device comprising of polymeric substrate, such as amatrix for example, that is used as the device body, and drugs, and insome cases additional scaffolding materials, such as metals oradditional polymers, and materials to enhance visibility and imaging. Animplantable delivery device can be advantageous in providing releaselocally and over a prolonged period, where drug is released directly tothe extracellular matrix (ECM) of the diseased area such as tumor,inflammation, degeneration or for symptomatic objectives, or to injuredsmooth muscle cells, or for prevention. One kind of drug is RNA, asdisclosed above, and this system may be used/and or adapted to theRNA-targeting system of the present invention. The modes of implantationin some embodiments are existing implantation procedures that aredeveloped and used today for other treatments, including brachytherapyand needle biopsy. In such cases the dimensions of the new implantdescribed in this invention are similar to the original implant.Typically a few devices are implanted during the same treatmentprocedure.

As described in US Patent Publication 20110195123, there is provided adrug delivery implantable or insertable system, including systemsapplicable to a cavity such as the abdominal cavity and/or any othertype of administration in which the drug delivery system is not anchoredor attached, comprising a biostable and/or degradable and/orbioabsorbable polymeric substrate, which may for example optionally be amatrix. It should be noted that the term “insertion” also includesimplantation. The drug delivery system is preferably implemented as a“Loder” as described in US Patent Publication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system as described in US Patent Publication20110195123 is optionally associated with sensing and/or activationappliances that are operated at and/or after implantation of the device,by non and/or minimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear—auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises an RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the RNA-targeting system of the presentinvention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the RNA-targeting system of the present invention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the RNA-targeting system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the RNA-targeting system of the presentinvention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Patient-Specific Screening Methods

An RNA-targeting system that targets RNA, e.g., trinucleotide repeatscan be used to screen patients or patent samples for the presence ofsuch repeats. The repeats can be the target of the RNA of theRNA-targeting system, and if there is binding thereto by theRNA-targeting system, that binding can be detected, to thereby indicatethat such a repeat is present. Thus, an RNA-targeting system can be usedto screen patients or patient samples for the presence of the repeat.The patient can then be administered suitable compound(s) to address thecondition; or, can be administered an RNA-targeting system to bind toand cause insertion, deletion or mutation and alleviate the condition.

Nucleic Acids, Amino Acids and Proteins, Regulatory Sequences, Vectors,Etc

Nucleic acids, amino acids and proteins: The invention uses nucleicacids to bind target RNA sequences. This is advantageous as nucleicacids are much easier and cheaper to produce than proteins, and thespecificity can be varied according to the length of the stretch wherehomology is sought. Complex 3-D positioning of multiple fingers, forexample is not required. The terms “polynucleotide”, “nucleotide”,“nucleotide sequence”, “nucleic acid” and “oligonucleotide” are usedinterchangeably. They refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three dimensional structure, andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: coding or non-coding regionsof a gene or gene fragment, loci (locus) defined from linkage analysis,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

In aspects of the invention the term “RNA-targeting guide RNA”, refersto the polynucleotide sequence comprising the RNA-targeting tracrsequence and the RNA-targeting sca sequence.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick base pairing or other non-traditional types. Apercent complementarity indicates the percentage of residues in anucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part I, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y. Where reference is made to apolynucleotide sequence, then complementary or partially complementarysequences are also envisaged. These are preferably capable ofhybridising to the reference sequence under highly stringent conditions.Generally, in order to maximize the hybridization rate, relativelylow-stringency hybridization conditions are selected: about 20 to 25° C.lower than the thermal melting point (T_(m)). The T_(m) is thetemperature at which 50% of specific target sequence hybridizes to aperfectly complementary probe in solution at a defined ionic strengthand pH. Generally, in order to require at least about 85% nucleotidecomplementarity of hybridized sequences, highly stringent washingconditions are selected to be about 5 to 15° C. lower than the T_(m). Inorder to require at least about 70% nucleotide complementarity ofhybridized sequences, moderately-stringent washing conditions areselected to be about 15 to 30° C. lower than the T_(m). Highlypermissive (very low stringency) washing conditions may be as low as 50°C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5 SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into and mRNAor other RNA transcript) and/or the process by which a transcribed mRNAis subsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

As used herein, the term “domain” or “protein domain” refers to a partof a protein sequence that may exist and function independently of therest of the protein chain. As described in aspects of the invention,sequence identity is related to sequence homology. Homology comparisonsmay be conducted by eye, or more usually, with the aid of readilyavailable sequence comparison programs. These commercially availablecomputer programs may calculate percent (%) homology between two or moresequences and may also calculate the sequence identity shared by two ormore amino acid or nucleic acid sequences. In some preferredembodiments, the capping region of the dTALEs described herein havesequences that are at least 95% identical or share identity to thecapping region amino acid sequences provided herein. Sequence homologiesmay be generated by any of a number of computer programs known in theart, for example BLAST or FASTA, etc. A suitable computer program forcarrying out such an alignment is the GCG Wisconsin Bestfit package(University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic AcidsResearch 12:387). Examples of other software than may perform sequencecomparisons include, but are not limited to, the BLAST package (seeAusubel et al., 1999 ibid—Chapter 18), FASTA (Atschul et al., 1990, J.Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. BothBLAST and FASTA are available for offline and online searching (seeAusubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferredto use the GCG Bestfit program. Percentage (%) sequence homology may becalculated over contiguous sequences, i.e., one sequence is aligned withthe other sequence and each amino acid or nucleotide in one sequence isdirectly compared with the corresponding amino acid or nucleotide in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues. Although this is a very simple andconsistent method, it fails to take into consideration that, forexample, in an otherwise identical pair of sequences, one insertion ordeletion may cause the following amino acid residues to be put out ofalignment, thus potentially resulting in a large reduction in % homologywhen a global alignment is performed. Consequently, most sequencecomparison methods are designed to produce optimal alignments that takeinto consideration possible insertions and deletions without undulypenalizing the overall homology or identity score. This is achieved byinserting “gaps” in the sequence alignment to try to maximize localhomology or identity. However, these more complex methods assign “gappenalties” to each gap that occurs in the alignment so that, for thesame number of identical amino acids, a sequence alignment with as fewgaps as possible—reflecting higher relatedness between the two comparedsequences—may achieve a higher score than one with many gaps. “Affinitygap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties may, of course, produce optimized alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example, when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension. Calculation of maximum % homologytherefore first requires the production of an optimal alignment, takinginto consideration gap penalties. A suitable computer program forcarrying out such an alignment is the GCG Wisconsin Bestfit package(Devereux et al., 1984 Nuc. Acids Research 12 p387). Examples of othersoftware than may perform sequence comparisons include, but are notlimited to, the BLAST package (see Ausubel et al., 1999Short Protocolsin Molecular Biology, 4^(th) Ed.—Chapter 18), FASTA (Altschul et al.,1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools.Both BLAST and FASTA are available for offline and online searching (seeAusubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58to 7-60). However, for some applications, it is preferred to use the GCGBestfit program. A new tool, called BLAST 2 Sequences is also availablefor comparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Compul. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyriylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g., circular); nucleic acid molecules thatcomprise DNA, RNA, or both; and other varieties of polynucleotides knownin the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for RNA-targetingCas protein (such as FnCas9) and RNA-targeting guide RNA. Bicistronicexpression vectors for RNA-targeting Cas protein (such as FnCas9) andRNA-targeting guide RNA are preferred. In general and particularly inthis embodiment RNA-targeting Cas protein (such as FnCas9) is preferablydriven by the CBh promoter. The RNA-targeting guide RNA may preferablybe driven by a Pol III promoter, such as a U6 promoter. Ideally the twoare combined. In practicing any of the methods disclosed herein, asuitable vector can be introduced to a cell or an embryo via one or moremethods known in the art, including without limitation, microinjection,electroporation, sonoporation, biolistics, calcium phosphate-mediatedtransfection, cationic transfection, liposome transfection, dendrimertransfection, heat shock transfection, nucleofection transfection,magnetofection, lipofection, impalefection, optical transfection,proprietary agent-enhanced uptake of nucleic acids, and delivery vialiposomes, immunoliposomes, virosomes, or artificial virions. In somemethods, the vector is introduced into an embryo by microinjection. Thevector or vectors may be microinjected into the nucleus or the cytoplasmof the embryo. In some methods, the vector or vectors may be introducedinto a cell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the 3-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1a promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).With regards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.,nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerivisae include pYepSec1(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomvces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Slaphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma.Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwninia, Fscherichia, Legionella, MAethylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia.Treponema, and Thermotoga.

In general, “RNA-targeting system” as used in the present applicationrefers collectively to transcripts and other elements involved in theexpression of or directing the activity of RNA-targetingCRISPR-associated (“Cas”) genes, including sequences encoding anRNA-targeting Cas protein and an RNA-targeting guide RNA (comprising anRNA-targeting small CRISPR/Cas system associated RNA (scaRNA) sequenceand an RNA-targeting trans-activating CRISPR/Cas system RNA (tracrRNA)sequence), or other sequences and transcripts from an RNA-targetingCRISPR locus. In some embodiments, one or more elements of anRNA-targeting system are derived from a type 1, type II, or type IIIRNA-targeting CRISPR system. In some embodiments, one or more elementsof an RNA-targeting system is derived from a particular organismcomprising an endogenous RNA-targeting CRISPR system, such asFrarcisella novicida. In general, an RNA-targeting system ischaracterized by elements that promote the formation of an RNA-targetingcomplex at the site of a target sequence. In the context of formation ofan RNA-targeting complex, “target sequence” refers to a sequence towhich a guide sequence is designed to have complementarity, wherehybridization between a target sequence and an RNA-targeting guide RNApromotes the formation of an RNA-targeting complex. Full complementarityis not necessarily required, provided there is sufficientcomplementarity to cause hybridization and promote formation of anRNA-targeting complex. A target sequence may comprise RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, the targetsequence may be within an organelle of a eukaryotic cell, for example,mitochondrion or chloroplast. A sequence or template that may be usedfor recombination into the targeted locus comprising the targetsequences is referred to as an “editing template” or “editing RNA” or“editing sequence”. In aspects of the invention, an exogenous templateRNA may be referred to as an editing template. In an aspect of theinvention the recombination is homologous recombination.

Typically, in the context of an endogenous RNA-targeting system,formation of an RNA-targeting complex (comprising an RNA-targeting guideRNA hybridized to a target sequence and complexed with one or moreRNA-targeting Cas proteins) results in cleavage of one or both RNAstrands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the target sequence. In some embodiments, oneor more vectors driving expression of one or more elements of anRNA-targeting system are introduced into a host cell such thatexpression of the elements of the RNA-targeting system direct formationof an RNA-targeting complex at one or more target sites. For example, anRNA-targeting Cas enzyme and an RNA-targeting guide RNA could each beoperably linked to separate regulatory elements on separate vectors.Alternatively, two or more of the elements expressed from the same ordifferent regulatory elements, may be combined in a single vector, withone or more additional vectors providing any components of theRNA-targeting system not included in the first vector. RNA-targetingsystem elements that are combined in a single vector may be arranged inany suitable orientation, such as one element located 5′ with respect to(“upstream” of) or 3′ with respect to (“downstream” of) a secondelement. The coding sequence of one element may be located on the sameor opposite strand of the coding sequence of a second element, andoriented in the same or opposite direction. In some embodiments, asingle promoter drives expression of a transcript encoding anRNA-targeting Cas protein and an RNA-targeting guide RNA embedded withinone or more intron sequences (e.g. each in a different intron, two ormore in at least one intron, or all in a single intron). In someembodiments, the RNA-targeting Cas protein and RNA-targeting guide RNAare operably linked to and expressed from the same promoter.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof an RNA-targeting complex to the target sequence. In some embodiments,the degree of complementarity between a guide sequence and itscorresponding target sequence, when optimally aligned using a suitablealignment algorithm, is about or more than about 50%, 60%, 75%, 80%,85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determinedwith the use of any suitable algorithm for aligning sequences,non-limiting example of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). In some embodiments, a guide sequence is about ormore than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotidesin length. In some embodiments, a guide sequence is less than about 75,50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Theability of a guide sequence to direct sequence-specific binding of anRNA-targeting complex to a target sequence may be assessed by anysuitable assay. For example, the components of an RNA-targeting systemsufficient to form an RNA-targeting complex, including the guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of the RNA-targeting CRISPR sequence, followedby an assessment of preferential cleavage within the target sequence,such as by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of an RNA-targeting complex,including the guide sequence to be tested and a control guide sequencedifferent from the test guide sequence, and comparing binding or rate ofcleavage at the target sequence between the test and control guidesequence reactions. Other assays are possible, and will occur to thoseskilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a gene transcriptor mRNA.

In some embodiments, the target sequence is a sequence within a genomeof a cell.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. TBA (attorney docket 44790.11.2022; Broad ReferenceBI-2013/004A); incorporated herein by reference.

In some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by an RNA-targetingCas protein as a part of an RNA-targeting complex. A templatepolynucleotide may be of any suitable length, such as about or more thanabout 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or morenucleotides in length. In some embodiments, the template polynucleotideis complementary to a portion of a polynucleotide comprising the targetsequence. When optimally aligned, a template polynucleotide mightoverlap with one or more nucleotides of a target sequences (e.g. aboutor more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100 or more nucleotides). In some embodiments, when a templatesequence and a polynucleotide comprising a target sequence are optimallyaligned, the nearest nucleotide of the template polynucleotide is withinabout 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000,10000, or more nucleotides from the target sequence.

In some embodiments, the RNA-targeting Cas protein is part of a fusionprotein comprising one or more heterologous protein domains (e.g., aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the RNA-targeting Cas protein). An RNA-targeting Cas proteinfusion protein may comprise any additional protein sequence, andoptionally a linker sequence between any two domains. Examples ofprotein domains that may be fused to an RNA-targeting Cas proteininclude, without limitation, epitope tags, reporter gene sequences, andprotein domains having one or more of the following activities:methylase activity, demethylase activity, transcription activationactivity, transcription repression activity, transcription releasefactor activity, histone modification activity, RNA cleavage activityand nucleic acid binding activity. Non-limiting examples of epitope tagsinclude histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Examples of reporter genes include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP).An RNA-targeting Cas protein may be fused to a gene sequence encoding aprotein or a fragment of a protein that bind DNA molecules or bind othercellular molecules, including but not limited to maltose binding protein(MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA bindingdomain fusions, and herpes simplex virus (HSV) BP16 protein fusions.Additional domains that may form part of a fusion protein comprising anRNA-targeting Cas protein are described in US20110059502, incorporatedherein by reference. In some embodiments, a tagged RNA-targeting Casprotein is used to identify the location of a target sequence.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, an RNA-targeting Cas protein in combination with (andoptionally complexed with) an RNA-targeting guide RNA is delivered to acell. Conventional viral and non-viral based gene transfer methods canbe used to introduce nucleic acids in mammalian cells or target tissues.Such methods can be used to administer nucleic acids encoding componentsof an RNA-targeting system to cells in culture, or in a host organism.Non-viral vector delivery systems include DNA plasmids, RNA (e.g. atranscript of a vector described herein), naked nucleic acid, andnucleic acid complexed with a delivery vehicle, such as a liposome.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and Immunology,Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ² cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells. WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of an RNA-targeting system as described herein (such as bytransient transfection of one or more vectors, or transfection withRNA), and modified through the activity of an RNA-targeting complex, isused to establish a new cell line comprising cells containing themodification but lacking any other exogenous sequence. In someembodiments, cells transiently or non-transiently transfected with oneor more vectors described herein, or cell lines derived from such cellsare used in assessing one or more test compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing an RNA-targeting complex to bind to the targetpolynucleotide to effect cleavage of said target polynucleotide therebymodifying the target polynucleotide, wherein the RNA-targeting complexcomprises an RNA-targeting Cas protein complexed with an RNA-targetingguide RNA hybridized to a target sequence within said targetpolynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing an RNA-targeting complex to bind to thepolynucleotide such that said binding results in increased or decreasedexpression of said polynucleotide; wherein the RNA-targeting complexcomprises an RNA-targeting Cas protein complexed with an RNA-targetingguide RNA hybridized to a target sequence within said polynucleotide.

With recent advances in crop genomics, the ability to use RNA-targetingsystems to perform efficient and cost effective gene editing andmanipulation will allow the rapid selection and comparison of single andmultiplexed genetic manipulations to transform such genomes for improvedproduction and enhanced traits. In this regard reference is made to USpatents and publications: U.S. Pat. No. 6,603,061—Agrobacterium-MediatedPlant Transformation Method; U.S. Pat. No. 7,868,149—Plant GenomeSequences and Uses Thereof and US 2009/0100536—Transgenic Plants withEnhanced Agronomic Traits, all the contents and disclosure of each ofwhich are herein incorporated by reference in their entirety. In thepractice of the invention, the contents and disclosure of Morrell et al“Crop genomics:advances and applications” Nat Rev Genet. 2011 Dec. 29;13(2):85-96 are also herein incorporated by reference in their entirety.In an advantageous embodiment of the invention, the RNA-targeting systemis used to engineer microalgae (Example 15). Accordingly, referenceherein to animal cells may also apply, mutatis mutandis, to plant cellsunless otherwise apparent.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Modifying a Target

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal or plant (includingmicro-algae), and modifying the cell or cells. Culturing may occur atany stage ex vivo. The cell or cells may even be re-introduced into thenon-human animal or plant. For re-introduced cells it is particularlypreferred that the cells are stem cells.

In some embodiments, the method comprises allowing an RNA-targetingcomplex to bind to the target polynucleotide to effect cleavage of saidtarget polynucleotide thereby modifying the target polynucleotide,wherein the RNA-targeting complex comprises an RNA-targeting Cas proteincomplexed with an RNA-targeting guide RNA hybridized or hybridizable toa target sequence within said target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing an RNA-targeting complex to bind to thepolynucleotide such that said binding results in increased or decreasedexpression of said polynucleotide; wherein the RNA-targeting complexcomprises an RNA-targeting Cas protein complexed with an RNA-targetingguide RNA hybridized or hybridizable to a target sequence within saidpolynucleotide. Similar considerations and conditions apply as above formethods of modifying a target polynucleotide. In fact, these sampling,culturing and re-introduction options apply across the aspects of thepresent invention.

Indeed, in any aspect of the invention, the RNA-targeting complex maycomprise an RNA-targeting Cas protein complexed with an RNA-targetingguide RNA hybridized or hybridizable to a target sequence. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide.

As described herein elsewhere, it will be apparent that in certainembodiments “modified”, “altered”, “manipulated” or like termscorresponds to alterations of target loci such as the activation orrepression of the transcription of a gene, methylation or demethylationof CpG sites and the like, which may not require point mutations,deletions, substitutions, or insertions of one or more nucleotides attarget loci. Furthermore as described herein elsewhere, it will also beapparent that reference to a CRISPR-Cas enzyme, including anRNA-targeting Cas protein, as “altering” or “modifying” or“manipulating” one or more target polynucleotide loci encompasses directalteration or modification, e.g. via the catalytic activity of theenzyme itself but also indirect alteration or modification, e.g. via acatalytic activity associated with the CRISPR-Cas enzyme or anRNA-targeting Cas protein, such as via a heterologous functional domain,e.g. a transcriptional activation domain or e.g. via a catalyticactivity of one or more heterologous functional domains associated withthe guide RNA via a protein-binding aptamer, e.g. a transcriptionalactivation domain. In addition, as it will be appreciated it is intendedthat the one or more target polynucleotide loci which are “altered” or“modified” by the action of the CRISPR-Cas enzyme or RNA-targeting Casprotein may be comprised in or adjacent the polynucleotide sequencecomplementary to the guide sequence portion of a guide RNA, e.g. inembodiments wherein the alteration or modification is effected by thecatalytic activity of the CRISPR-Cas enzyme or RNA-targeting Cas proteinitself, e.g. cleavage of DNA by the nuclease activity of the CRISPR-Casenzyme or Cas protein. However, also encompassed are embodiments whereinone or more target loci to be “altered” or “modified” are at a locationdistinct from the sequence complementary to the guide sequence portionof the guide RNA, e.g. in embodiments wherein the alteration ormodification is effected via a heterologous functional domain associatedwith the CRISPR-Cas enzyme or Cas protein and/or guide RNA, e.g.activation or repression of the transcription of a gene. As such,“alteration” or “modification” (or analogous terms) of a target locusmeans via direct or indirect action of the CRISPR-Cas enzyme or Casprotein, and furthermore the “target locus” to be altered or modifiedand the “target sequence” which is complementary to the guide sequenceportion of the guide RNA may or may not be the same.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthii Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

Kits

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system as taught herein andinstructions for using the kit. Elements may be provide individually orin combinations, and may be provided in any suitable container, such asa vial, a bottle, or a tube. In some embodiments, the kit includesinstructions in one or more languages, for example in more than onelanguage. The instructions may be specific to the applications andmethods described herein.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.,in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide. In some embodiments, the kitcomprises one or more of the vectors and/or one or more of thepolynucleotides described herein. The kit may advantageously allows toprovide all elements of the systems of the invention.

In one aspect, the invention provides methods for using one or moreelements of an RNA-targeting system. The RNA-targeting complex of theinvention provides an effective means for modifying a targetpolynucleotide. The RNA-targeting complex of the invention has a widevariety of utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) a target polynucleotide in amultiplicity of cell types. As such the RNA-targeting complex of theinvention has a broad spectrum of applications in, e.g., gene therapy,drug screening, disease diagnosis, and prognosis.

The crystals of the RNA-targeting complex such as the RNA-targetingFnCas9 complex can be obtained by techniques of protein crystallography,including batch, liquid bridge, dialysis, vapor diffusion and hangingdrop methods. Generally, the crystals of the invention are grown bydissolving substantially pure RNA-targeting complex and a nucleic acidmolecule to which it binds in an aqueous buffer containing a precipitantat a concentration just below that necessary to precipitate. Water isremoved by controlled evaporation to produce precipitating conditions,which are maintained until crystal growth ceases. The crystal structureinformation is described in U.S. provisional applications 61/915,251filed Dec. 12, 2013, 61/930,214 filed on Jan. 22, 2014, 61/980,012 filedApr. 15, 2014; and Nishimasu et al, “Crystal Structure of Cas9 inComplex with Guide RNA and Target DNA,” Cell 156(5):935-949, DOI:accessible at dx.doi.org/10.1016/j.cell.2014.02.001 (2014), each and allof which are incorporated herein by reference.

Uses of the Crystals, Crystal Structure and Atomic StructureCo-Ordinates: The crystals of the RNA-targeting complex such as theRNA-targeting FnCas9 complex, and particularly the atomic structureco-ordinates obtained therefrom, have a wide variety of uses. Thecrystals and structure co-ordinates are particularly useful foridentifying compounds (nucleic acid molecules) that bind toRNA-targeting complexes such as the RNA-targeting FnCas9 complex, andRNA-targeting complexes that can bind to particular compounds (nucleicacid molecules). Thus, the structure co-ordinates described herein canbe used as phasing models in determining the crystal structures ofadditional synthetic or mutated RNA-targeting complexes such as theRNA-targeting FnCas9 complex, FnCas9, nickases, binding domains. Theprovision of the crystal structure of RNA-targeting complexes such asthe RNA-targeting FnCas9 complex complexed with a nucleic acid moleculeas applied in conjunction with the herein teachings provides the skilledartisan with a detailed insight into the mechanisms of action ofRNA-targeting complexes such as the RNA-targeting FnCas9 complex. Thisinsight provides a means to design modified RNA-targeting complexes,such as by attaching thereto a functional group, such as a repressor oractivator. While one can attach a functional group such as a repressoror activator to the N or C terminal of RNA-targeting complexes, thecrystal structure demonstrates that the N terminal seems obscured orhidden, whereas the C terminal is more available for a functional groupsuch as repressor or activator. Attachment can be via a linker, e.g., aflexible glycine-serine (GlyGlyGlySer (SEQ ID NO: 23)) or (GGGS)₃ (SEQID NO: 28) or a rigid alpha-helical linker such as(Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO: 29). In addition to the flexibleloop there is also a nuclease or H3 region, an H2 region and a helicalregion. By “helix” or “helical”, is meant a helix as known in the art,including, but not limited to an alpha-helix. Additionally, the termhelix or helical may also be used to indicate a c-terminal helicalelement with an N-terminal turn.

The provision of the crystal structure of RNA-targeting complex such asRNA-targeting FnCas9 complex complexed with a nucleic acid moleculeallows a novel approach for drug or compound discovery, identification,and design for compounds that can bind to RNA-targeting complexes andthus the invention provides tools useful in diagnosis, treatment, orprevention of conditions or diseases of multicellular organisms, e.g.,algae, plants, invertebrates, fish, amphibians, reptiles, avians,mammals; for example domesticated plants, animals (e.g., productionanimals such as swine, bovine, chicken; companion animal such asfelines, canines, rodents (rabbit, gerbil, hamster); laboratory animalssuch as mouse, rat), and humans.

In any event, the determination of the three-dimensional structure ofRNA-targeting complexes such as FnCas9 complex provides a basis for thedesign of new and specific nucleic acid molecules that bind toCRISPR-Cas 9 (e.g., FnCas9), as well as the design of new RNA-targetingCRISPR-Cas9 systems, such as by way of modification of the RNA-targetingCRISPR-Cas9 system to bind to various nucleic acid molecules, by way ofmodification of the RNA-targeting CRISPR-Cas9 system to have linkedthereto to any one or more of various functional groups that mayinteract with each other, with the RNA-targeting CRISPR-Cas9 (e.g., aninducible system that provides for self-activation and/orself-termination of function), with the nucleic acid molecule nucleicacid molecules (e.g., the functional group may be a regulatory orfunctional domain which may be selected from the group comprising,consisting essentially of, or consisting of a transcriptional repressor,a transcriptional activator, a nuclease domain, a DNA methyltransferase, a protein acetyltransferase, a protein deacetylase, aprotein methyltransferase, a protein deaminase, a protein kinase, and aprotein phosphatase; and, in some aspects, the functional domain is anepigenetic regulator, see, e.g., Zhang et al., U.S. Pat. No. 8,507,272,and it is again mentioned that it and all documents cited herein and allappln cited documents are hereby incorporated herein by reference), byway of modification of Cas9, by way of novel nickases). Indeed, theRNA-targeting CRISPR-Cas9 (FnCas9) crystal structure has a multitude ofuses. For example, from knowing the three-dimensional structure ofRNA-targeting CRISPR-Cas9 (FnCas9) crystal structure, computer modellingprograms may be used to design or identify different molecules expectedto interact with possible or confirmed sites such as binding sites orother structural or functional features of the RNA-targeting CRISPR-Cas9system (e.g., FnCas9). Compound that potentially bind (“binder”) can beexamined through the use of computer modeling using a docking program.Docking programs are known; for example GRAM, DOCK or AUTODOCK (seeWalters et al. Drug Discovery Today, vol. 3, no. 4 (1998), 160-178, andDunbrack et al. Folding and Design 2 (1997), 27-42). This procedure caninclude computer fitting of potential binders ascertain how well theshape and the chemical structure of the potential binder will bind to anRNA-targeting CRISPR-Cas9 system (e.g., FnCas9). Computer-assisted,manual examination of the active site or binding site of anRNA-targeting CRISPR-Cas9 system (e.g., FnCas9) may be performed.Programs such as GRID (P. Goodford, J. Med. Chem, 1985, 28, 849-57)—aprogram that determines probable interaction sites between moleculeswith various functional groups—may also be used to analyze the activesite or binding site to predict partial structures of binding compounds.Computer programs can be employed to estimate the attraction, repulsionor steric hindrance of the two binding partners, e.g., RNA-targetingCRISPR-Cas9 system (e.g., FnCas9) and a candidate nucleic acid moleculeor a nucleic acid molecule and a candidate RNA-targeting CRISPR-Cas9system (e.g., FnCas9); and the RNA-targeting CRISPR-Cas9 crystalstructure (FnCas9) enables such methods. Generally, the tighter the fit,the fewer the steric hindrances, and the greater the attractive forces,the more potent the potential binder, since these properties areconsistent with a tighter binding constant. Furthermore, the morespecificity in the design of a candidate RNA-targeting CRISPR-Cas9system (e.g., FnCas9), the more likely it is that it will not interactwith off-target molecules as well. Also, “wet” methods are enabled bythe instant invention. For example, in an aspect, the invention providesfor a method for determining the structure of a binder (e.g., targetnucleic acid molecule) of a candidate CRISPR-Cas9 system (e.g., FnCas9)bound to the candidate RNA-targeting CRISPR-Cas9 system (e.g., FnCas9),said method comprising, (a) providing a first crystal of a candidateRNA-targeting CRISPR-Cas9 system (FnCas9) according to the invention ora second crystal of a candidate RNA-targeting CRISPR-Cas9 system (e.g.,FnCas9), (b) contacting the first crystal or second crystal with saidbinder under conditions whereby a complex may form; and (c) determiningthe structure of said candidate (e.g., CRISPR-Cas9 system (e.g., FnCas9)or CRISPR-Cas9 system (FnCas9) complex. The second crystal may haveessentially the same coordinates discussed herein, however due to minoralterations in RNA-targeting CRISPR-Cas9 system (e.g., from the Cas9 ofsuch a system being e.g., FnCas9 versus being FnCas9), wherein “e.g.,FnCas9” indicates that the Cas9 is a Cas9 and can be of or derived fromFrancisella novicida or an ortholog thereof), the crystal may form in adifferent space group.

The invention further involves, in place of or in addition to “insilico” methods, other “wet” methods, including high throughputscreening of a binder (e.g., target nucleic acid molecule) and acandidate RNA-targeting CRISPR-Cas9 system (e.g., FnCas9), or acandidate binder (e.g., target nucleic acid molecule) and anRNA-targeting CRISPR-Cas9 system (e.g., FnCas9), or a candidate binder(e.g., target nucleic acid molecule) and a candidate RNA-targetingCRISPR-Cas9 system (e.g., FnCas9) (the foregoing CRISPR-Cas9 system(s)with or without one or more functional group(s)), to select compoundswith binding activity. Those pairs of binder and RNA-targetingCRISPR-Cas9 system which show binding activity may be selected andfurther crystallized with the RNA-targeting CRISPR-Cas9 crystal having astructure herein, e.g., by co-crystallization or by soaking, for X-rayanalysis. The resulting X-ray structure may be compared with that of theCas9 Crystal Structure for a variety of purposes, e.g., for areas ofoverlap. Having designed, identified, or selected possible pairs ofbinder and RNA-targeting CRISPR-Cas9 system by determining those whichhave favorable fitting properties, e.g., predicted strong attractionbased on the pairs of binder and RNA-targeting CRISPR-Cas9 crystalstructure data herein, these possible pairs can then be screened by“wet” methods for activity. Consequently, in an aspect the invention caninvolve: obtaining or synthesizing the possible pairs, and contacting abinder (e.g., target nucleic acid molecule) and a candidateRNA-targeting CRISPR-Cas9 system (e.g., FnCas9), or a candidate binder(e.g., target nucleic acid molecule) and an RNA-targeting CRISPR-Cas9system (e.g., FnCas9), or a candidate binder (e.g., target nucleic acidmolecule) and a candidate RNA-targeting CRISPR-Cas9 system (e.g.,FnCas9) (the foregoing CRISPR-Cas9 system(s) with or without one or morefunctional group(s)) to determine ability to bind. In the latter step,the contacting is advantageously under conditions to determine function.Instead of, or in addition to, performing such an assay, the inventionmay comprise: obtaining or synthesizing complex(es) from said contactingand analyzing the complex(es), e.g., by X-ray diffraction or NMR orother means, to determine the ability to bind or interact. Detailedstructural information can then be obtained about the binding, and inlight of this information, adjustments can be made to the structure orfunctionality of a candidate RNA-targeting CRISPR-Cas9 system orcomponents thereof. These steps may be repeated and re-repeated asnecessary. Alternatively or additionally, potential RNA-targetingCRISPR-Cas9 systems from or in the foregoing methods can be with nucleicacid molecules in vivo, including without limitation by way ofadministration to an organism (including non-human animal and human) toascertain or confirm function, including whether a desired outcome(e.g., reduction of symptoms, treatment) results therefrom.

The invention further involves a method of determining three dimensionalstructures of RNA-targeting systems or complex(es) of unknown structureby using the structural co-ordinates of the Cas9 Crystal Structure. Forexample, if X-ray crystallographic or NMR spectroscopic data areprovided for an RNA-targeting system or complex of unknown crystalstructure, the structure of an RNA-targeting complex may be used tointerpret that data to provide a likely structure for the unknown systemor complex by such techniques as by phase modeling in the case of X-raycrystallography. Thus, an inventive method can comprise: aligning arepresentation of the RNA-targeting system or complex having an unknowncrystal structure with an analogous representation of the RNA-targetingCRISPR-cas(9) system and complex of the crystal structure herein tomatch homologous or analogous regions (e.g., homologous or analogoussequences); modeling the structure of the matched homologous oranalogous regions (e.g., sequences) of the RNA-targeting system orcomplex of unknown crystal structure based on the structure of the Cas9Crystal Structure of the corresponding regions (e.g., sequences); and,determining a conformation (e.g., taking into consideration favorableinteractions should be formed so that a low energy conformation isformed) for the unknown crystal structure which substantially preservesthe structure of said matched homologous regions. “Homologous regions”describes, for example as to amino acids, amino acid residues in twosequences that are identical or have similar, e.g., aliphatic, aromatic,polar, negatively charged, or positively charged, side-chain chemicalgroups. Homologous regions as or nucleic acid molecules can include atleast 85% or 86% or 87% or 88% or 89% or 90% or 91% or 92% or 93% or 94%or 95% or 96% or 97% or 98% or 99% homology or identity. Identical andsimilar regions are sometimes described as being respectively“invariant” and “conserved” by those skilled in the art. Homologymodeling is a technique that is well known to those skilled in the art(see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell et al. EurJ Biochem vol 172 (1988), 513). The computer representation of theconserved regions of the RNA-targeting CRISPR-Cas9 crystal structure andthose of an RNA-targeting system of unknown crystal structure aid in theprediction and determination of the crystal structure of theRNA-targeting system of unknown crystal structure.

Further still, the aspects of the invention which employ the CRISPR-Cas9crystal structure in silico may be equally applied to new RNA-targetingcrystal structures divined by using the herein-referenced CRISPR-Cas9crystal structure. In this fashion, a library of RNA-targeting complexcrystal structures can be obtained. Rational RNA-targeting system designis thus provided by the instant invention. For instance, havingdetermined a conformation or crystal structure of an RNA-targetingsystem or complex, by the methods described herein, such a conformationmay be used in a computer-based method herein for determining theconformation or crystal structure of other RNA-targeting systems orcomplexes whose crystal structures are yet unknown. Data from all ofthese crystal structures can be in a database, and the herein methodscan be more robust by having herein comparisons involving the hereincrystal structure or portions thereof be with respect to one or morecrystal structures in the library. The invention further providessystems, such as computer systems, intended to generate structuresand/or perform rational design of an RNA-targeting system or complex.The system can contain: atomic co-ordinate data according to theherein-referenced Crystal Structure or be derived therefrom e.g., bymodeling, said data defining the three-dimensional structure of anRNA-targeting system or complex or at least one domain or sub-domainthereof, or structure factor data therefor, said structure factor databeing derivable from the atomic co-ordinate data of theherein-referenced Crystal Structure. The invention also involvescomputer readable media with: atomic co-ordinate data according to theherein-referenced Crystal Structure or derived therefrom e.g., byhomology modeling, said data defining the three-dimensional structure ofan RNA-targeting system or complex or at least one domain or sub-domainthereof, or structure factor data therefor, said structure factor databeing derivable from the atomic co-ordinate data of theherein-referenced Crystal Structure. “Computer readable media” refers toany media which can be read and accessed directly by a computer, andincludes, but is not limited to: magnetic storage media; optical storagemedia; electrical storage media; cloud storage and hybrids of thesecategories. By providing such computer readable media, the atomicco-ordinate data can be routinely accessed for modeling or other “insilico” methods. The invention further comprehends methods of doingbusiness by providing access to such computer readable media, forinstance on a subscription basis, via the Internet or a globalcommunication/computer network; or, the computer system can be availableto a user, on a subscription basis. A “computer system” refers to thehardware means, software means and data storage means used to analyzethe atomic co-ordinate data of the present invention. The minimumhardware means of computer-based systems of the invention may comprise acentral processing unit (CPU), input means, output means, and datastorage means. Desirably, a display or monitor is provided to visualizestructure data. The invention further comprehends methods oftransmitting information obtained in any method or step thereofdescribed herein or any information described herein, e.g., viatelecommunications, telephone, mass communications, mass media,presentations, internet, email, etc. The crystal structures of theinvention can be analyzed to generate Fourier electron density map(s) ofRNA-targeting systems or complexes; advantageously, thethree-dimensional structure being as defined by the atomic co-ordinatedata according to the herein-referenced Crystal Structure. Fourierelectron density maps can be calculated based on X-ray diffractionpatterns. These maps can then be used to determine aspects of binding orother interactions. Electron density maps can be calculated using knownprograms such as those from the CCP4 computer package (CollaborativeComputing Project, No. 4. The CCP4 Suite: Programs for ProteinCrystallography, Acta Crystallographica, D50, 1994, 760-763). For mapvisualization and model building programs such as “QUANTA” (1994, SanDiego, Calif.: Molecular Simulations, Jones et al., Acta CrystallographyA47 (1991), 110-119) can be used.

The herein-referenced Crystal Structure gives atomic co-ordinate datafor a CRISPR-Cas9 (S. pyogenes), and lists each atom by a unique number;the chemical element and its position for each amino acid residue (asdetermined by electron density maps and antibody sequence comparisons),the amino acid residue in which the element is located, the chainidentifier, the number of the residue, co-ordinates (e.g., X, Y, Z)which define with respect to the crystallographic axes the atomicposition (in angstroms) of the respective atom, the occupancy of theatom in the respective position, “B”, isotropic displacement parameter(in angstroms²) which accounts for movement of the atom around itsatomic center, and atomic number.

In particular embodiments of the invention, the conformationalvariations in the crystal structures of the CRISPR-Cas9 system or ofcomponents of the CRISPR-Cas9 system provide important and criticalinformation about the flexibility or movement of protein structureregions relative to nucleotide (RNA or DNA) structure regions that maybe important for RNA-targeting system function. The structuralinformation provided for Cas9 (e.g., F. novicida Cas9) as the CRISPRenzyme in the present application may be used to further engineer andoptimize the RNA-targeting system and this may be extrapolated tointerrogate structure-function relationships in other CRISPR enzymesystems as well. In particular embodiments of the invention, the crystalstructure provides a critical step towards understanding the molecularmechanism of RNA-guided DNA targeting by Cas9. The structural andfunctional analyses herein provide a useful scaffold for rationalengineering of Cas9-based genome modulating technologies and may provideguidance as to Cas9-mediated recognition of PAM sequences on the targetDNA or mismatch tolerance between the sgRNA:DNA duplex. Aspects of theinvention also relate to truncation mutants, e.g., an FnCas9 truncationmutant may facilitate packaging of Cas9 into size-constrained viralvectors for in vivo and therapeutic applications. Similarly, engineeringof the PAM Interacting (PI) domain may allow programming of PAMspecificity, improve target site recognition fidelity, and increase theversatility of the Cas9 genome engineering platform. Cas9 proteins maybe engineered to alter their PAM specificity, for example as describedin Kleinstiver B P et al. Engineered CRISPR-Cas9 micleases with alteredPAM specficities. Nature. 2015 Jul. 23; 523(7561):481-5. doi:10.1038/nature14592.

The structural information provided herein allows for interrogation ofRNA-targeting guide RNA interaction with the target DNA and theRNA-targeting Cas protein (e.g., Cas9) permitting engineering oralteration of RNA-targeting guide RNA structure to optimizefunctionality of the entire RNA-targeting CRISPR-Cas system. Forexample, loops of the RNA-targeting guide RNA may be extended, withoutcolliding with the RNA-targeting Cas protein by the insertion ofdistinct RNA loop(s) or distinct sequence(s) that may recruit adaptorproteins that can bind to the distinct RNA loop(s) or distinctsequence(s). The adaptor proteins may include but are not limited toorthogonal RNA-binding protein/aptamer combinations that exist withinthe diversity of bacteriophage coat proteins. A list of such coatproteins includes, but is not limited to: Qβ, F2, GA, fr, JP501, M12,R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95,TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. These adaptorproteins or orthogonal RNA binding proteins can further recruit effectorproteins or fusions which comprise one or more functional domains. Insome embodiments, the functional domain may be selected from the groupcomprising, consisting essentially of, or consisting of: transposasedomain, integrase domain, recombinase domain, resolvase domain,invertase domain, protease domain, DNA methyltransferase domain, DNAhydroxylmethylase domain, DNA demethylase domain, histone acetylasedomain, histone deacetylases domain, nuclease domain, repressor domain,activator domain, nuclear-localization signal domains,transcription-regulatory protein (or transcription complex recruiting)domain, cellular uptake activity associated domain, nucleic acid bindingdomain, antibody presentation domain, histone modifying enzymes,recruiter of histone modifying enzymes; inhibitor of histone modifyingenzymes, histone methyltransferase, histone demethylase, histone kinase,histone phosphatase, histone ribosylase, histone deribosylase, histoneubiquitinase, histone deubiquitinase, histone biotinase and histone tailprotease.

In some preferred embodiments, the functional domain is atranscriptional activation domain, preferably VP64. In some embodiments,the functional domain is a transcription repression domain, preferablyKRAB. In some embodiments, the transcription repression domain is SID,or concatemers of SID (eg SID4X). In some embodiments, the functionaldomain is an epigenetic modifying domain, such that an epigeneticmodifying enzyme is provided. In some embodiments, the functional domainis an activation domain, which may be the P65 activation domain.

In one aspect surveyor analysis is used for identification of indelactivity/nuclease activity. In general survey analysis includesextraction of genomic DNA, PCR amplification of the genomic regionflanking the CRISPR target site, purification of products, re-annealingto enable heteroduplex formation. After re-annealing, products aretreated with SURVEYOR nuclease and SURVEYOR enhancer S (Transgenomics)following the manufacturer's recommended protocol. Analysis may beperformed with poly-acrylamide gels according to known methods.Quantification may be based on relative band intensities.

Crystallization of CRISPR-Cas9 and Characterization of Crystal Structure

The crystals of the Cas9 can be obtained by techniques of proteincrystallography, including batch, liquid bridge, dialysis, vapordiffusion and hanging drop methods. Generally, the crystals of theinvention are grown by dissolving substantially pure CRISPR-Cas9 and anucleic acid molecule to which it binds in an aqueous buffer containinga precipitant at a concentration just below that necessary toprecipitate. Water is removed by controlled evaporation to produceprecipitating conditions, which are maintained until crystal growthceases. The crystal structure information is described in U.S.provisional applications 61/915,251 filed Dec. 12, 2013, 61/930,214filed on Jan. 22, 2014, 61/980,012 filed Apr. 15, 2014; and Nishimasu etal, “Crystal Structure of Cas9 in Complex with Guide RNA and TargetDNA,” Cell 156(5):935-949, DOI: accessible atdx.doi.org/10.1016/j.cell.2014.02.001 (2014), and PCT/US14/70175; andNishimasu H et al, Crystal Structure of Staphylococcus aureus Cas9.Cell. 2015 Aug. 27; 162(5):1113-26. doi: 10.1016/j.cell.2015.08.007;each and all of which are incorporated herein by reference, and togetherare “herein cited materials” concerning the Cas9 crystal structure. Usesof the Crystals, Crystal Structure and Atomic Structure Co-Ordinates ofthe herein cited materials: The crystals of the Cas9, and particularlythe atomic structure co-ordinates obtained therefrom, have a widevariety of uses. The crystals and structure co-ordinates areparticularly useful for identifying compounds (nucleic acid molecules)that bind to CRISPR-Cas9, and CRISPR-Cas9s that can bind to particularcompounds (nucleic acid molecules). Thus, the structure co-ordinatesdescribed herein can be used as phasing models in determining thecrystal structures of additional synthetic or mutated CRISPR-Cas9s,Cas9s, nickases, binding domains. The provision of the crystal structureof CRISPR-Cas9 complexed with a nucleic acid molecule as applied inconjunction with the herein teachings provides the skilled artisan witha detailed insight into the mechanisms of action of CRISPR-Cas9. Thisinsight provides a means to design modified CRISPR-Cas9s, such as byattaching thereto a functional group, such as a repressor or activator.While one can attach a functional group such as a repressor or activatorto the N or C terminal of CRISPR-Cas9, the crystal structuredemonstrates that the N terminal seems obscured or hidden, whereas the Cterminal is more available for a functional group such as repressor oractivator. Moreover, the crystal structure demonstrates that there is aflexible loop between approximately CRISPR-Cas9 (S. pyogenes) residues534-676 which is suitable for attachment of a functional group such asan activator or repressor. Attachment can be via a linker, e.g., aflexible glycine-serine (GlyGlyGlySer (SEQ ID NO: 23)) or (GGGS)₃ (SEQID NO: 28) or a rigid alpha-helical linker such as(Ala(GluAlaAlaAlaLys)Ala) (SEQ ID NO: 29). In addition to the flexibleloop there is also a nuclease or H3 region, an H2 region and a helicalregion. By “helix” or “helical”, is meant a helix as known in the art,including, but not limited to an alpha-helix. Additionally, the termhelix or helical may also be used to indicate a c-terminal helicalelement with an N-terminal turn. The provision of the crystal structureof CRISPR-Cas9 complexed with a nucleic acid molecule allows a novelapproach for drug or compound discovery, identification, and design forcompounds that can bind to CRISPR-Cas9 and thus the invention providestools useful in diagnosis, treatment, or prevention of conditions ordiseases of multicellular organisms, e.g., algae, plants, invertebrates,fish, amphibians, reptiles, avians, mammals; for example domesticatedplants, animals (e.g., production animals such as swine, bovine,chicken; companion animal such as felines, canines, rodents (rabbit,gerbil, hamster); laboratory animals such as mouse, rat), and humans.The invention further involves, in place of or in addition to “insilico” methods, other “wet” methods, including high throughputscreening of a binder (e.g., target nucleic acid molecule) and acandidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidatebinder (e.g., target nucleic acid molecule) and a CRISPR-Cas9 system(e.g., S. pyogenes Cas9), or a candidate binder (e.g., target nucleicacid molecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenesCas9) (the foregoing CRISPR-Cas9 system(s) with or without one or morefunctional group(s)), to select compounds with binding activity. Thosepairs of binder and CRISPR-Cas9 system which show binding activity maybe selected and further crystallized with the CRISPR-Cas9 crystal havinga structure herein, e.g., by co-crystallization or by soaking, for X-rayanalysis. The resulting X-ray structure may be compared with that of theCas9 Crystal Structure for a variety of purposes, e.g., for areas ofoverlap. Having designed, identified, or selected possible pairs ofbinder and CRISPR-Cas9 system by determining those which have favorablefitting properties, e.g., predicted strong attraction based on the pairsof binder and CRISPR-Cas9 crystal structure data herein, these possiblepairs can then be screened by “wet” methods for activity. Consequently,in an aspect the invention can involve: obtaining or synthesizing thepossible pairs; and contacting a binder (e.g., target nucleic acidmolecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9),or a candidate binder (e.g., target nucleic acid molecule) and aCRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidate binder(e.g., target nucleic acid molecule) and a candidate CRISPR-Cas9 system(e.g., S. pyogenes Cas9) (the foregoing CRISPR-Cas9 system(s) with orwithout one or more functional group(s)) to determine ability to bind.In the latter step, the contacting is advantageously under conditions todetermine function. Instead of, or in addition to, performing such anassay, the invention may comprise: obtaining or synthesizing complex(es)from said contacting and analyzing the complex(es), e.g., by X-raydiffraction or NMR or other means, to determine the ability to bind orinteract. Detailed structural information can then be obtained about thebinding, and in light of this information, adjustments can be made tothe structure or functionality of a candidate CRISPR-Cas9 system orcomponents thereof. These steps may be repeated and re-repeated asnecessary. Alternatively or additionally, potential CRISPR-Cas9 systemsfrom or in the foregoing methods can be with nucleic acid molecules invivo, including without limitation by way of administration to anorganism (including non-human animal and human) to ascertain or confirmfunction, including whether a desired outcome (e.g., reduction ofsymptoms, treatment) results therefrom. If X-ray crystallographic or NMRspectroscopic data are provided for a CRISPR-cas system or complex ofunknown crystal structure, the structure of a CRISPR-Cas9 complex asdefined of the herein cited materials may be used to interpret that datato provide a likely structure for the unknown system or complex by suchtechniques as by phase modeling in the case of X-ray crystallography.Thus, from this disclosure and the knowledge in the art one can performa method comprising: aligning a representation of the CRISPR-cas systemor complex having an unknown crystal structure with an analogousrepresentation of the CRISPR-cas(9) system and complex of the crystalstructure herein to match homologous or analogous regions (e.g.,homologous or analogous sequences); modeling the structure of thematched homologous or analogous regions (e.g., sequences) of theCRISPR-cas system or complex of unknown crystal structure based on thestructure of the Cas9 Crystal Structure of the corresponding regions(e.g., sequences); and, determining a conformation (e.g. taking intoconsideration favorable interactions should be formed so that a lowenergy conformation is formed) for the unknown crystal structure whichsubstantially preserves the structure of said matched homologousregions. “Homologous regions” describes, for example as to amino acids,amino acid residues in two sequences that are identical or have similar,e.g., aliphatic, aromatic, polar, negatively charged, or positivelycharged, side-chain chemical groups. Homologous regions as ot nucleicacid molecules can include at least 85% or 86% or 87% or 88% or 89% or90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 990%homology or identity. Identical and similar regions are sometimesdescribed as being respectively “invariant” and “conserved” by thoseskilled in the art. Homology modeling is a technique that is well knownto those skilled in the art (see, e.g., Greer, Science vol. 228 (1985)1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513). Thecomputer representation of the conserved regions of the CRISPR-Cas9crystal structure and those of a CRISPR-Cas system of unknown crystalstructure aid in the prediction and determination of the crystalstructure of the CRISPR-cas system of unknown crystal structure. TheCRISPR-Cas9 crystal structure in silico may be equally applied to newCRISPR-cas crystal structures divined by using the teachings herein andthe herein cited materials concerning the CRISPR-Cas9 crystal structure.In this fashion, a library of CRISPR-Cas crystal structures can beobtained. Rational CRISPR-Cas system design is thus provided by theinstant invention. For instance, having obtained a CRISPR-Cas system orcomplex using teachings herein and determining a conformation or crystalstructure of a CRISPR-Cas system or complex, such a conformation may beused in a computer-based methods herein for determining the conformationor crystal structure of other CRISPR-Cas systems or complexes whosecrystal structures are yet unknown. Data from all of these crystalstructures can be in a database, and the herein methods can be morerobust by having herein comparisons involving the herein crystalstructure or portions thereof be with respect to one or more crystalstructures in the library. Thus, there is the provision of systems, suchas computer systems, intended to generate structures and/or performrational design of a CRISPR-Cas system or complex. The system cancontain: atomic co-ordinate data according to the herein cited materialsconcerning the Cas9 Crystal Structure or be derived therefrom e.g., bymodeling, said data defining the three-dimensional structure of aCRISPR-Cas system or complex or at least one domain or sub-domainthereof, or structure factor data therefor, said structure factor databeing derivable from the atomic co-ordinate data of theherein-referenced Crystal Structure. The invention also involvescomputer readable media with: atomic co-ordinate data according to theteachings herein e.g., by or from homology modeling, said data definingthe three-dimensional structure of a CRISPR-Cas system or complex or atleast one domain or sub-domain thereof, or structure factor datatherefor, said structure factor data being derivable from the atomicco-ordinate data of the herein-referenced Crystal Structure. “Computerreadable media” refers to any media which can be read and accesseddirectly by a computer, and includes, but is not limited to: magneticstorage media; optical storage media; electrical storage media; cloudstorage and hybrids of these categories. By providing such computerreadable media, the atomic co-ordinate data can be routinely accessedfor modeling or other “in silico” methods. The invention furthercomprehends methods of doing business by providing access to suchcomputer readable media, for instance on a subscription basis, via theInternet or a global communication/computer network; or, the computersystem can be available to a user, on a subscription basis. A “computersystem” refers to the hardware means, software means and data storagemeans used to analyze the atomic co-ordinate data of the presentinvention. The minimum hardware means of computer-based systems of theinvention may comprise a central processing unit (CPU), input means,output means, and data storage means. Desirably, a display or monitor isprovided to visualize structure data. The invention further comprehendsmethods of transmitting information obtained in any method or stepthereof described herein or any information described herein, e.g., viatelecommunications, telephone, mass communications, mass media,presentations, internet, email, etc. The crystal structures of theinvention can be analyzed to generate Fourier electron density map(s) ofCRISPR-Cas systems or complexes; advantageously, the three-dimensionalstructure being as defined by the atomic co-ordinate data according tothe herein-referenced Crystal Structure. Fourier electron density mapscan be calculated based on X-ray diffraction patterns. These maps canthen be used to determine aspects of binding or other interactions.Electron density maps can be calculated using known programs such asthose from the CCP4 computer package (Collaborative Computing Project,No. 4. The CCP4 Suite: Programs for Protein Crystallography, ActaCrystallographica, D50, 1994, 760-763). For map visualization and modelbuilding programs such as “QUANTA” (1994, San Diego, Calif.: MolecularSimulations, Jones et al., Acta Crystallography A47 (1991), 110-119) canbe used.

The herein cited materials concerning the Crystal Structure gives atomicco-ordinate data for a CRISPR-Cas9 (S. pyogenes), and lists each atom bya unique number; the chemical element and its position for each aminoacid residue (as determined by electron density maps and antibodysequence comparisons), the amino acid residue in which the element islocated, the chain identifier, the number of the residue, co-ordinates(e.g., X, Y, Z) which define with respect to the crystallographic axesthe atomic position (in angstroms) of the respective atom, the occupancyof the atom in the respective position, “B”, isotropic displacementparameter (in angstroms²) which accounts for movement of the atom aroundits atomic center, and atomic number. The conformational variations inthe crystal structures of the CRISPR-Cas9 system or of components of theCRISPR-Cas9 may provide important and critical information about theflexibility or movement of protein structure regions relative tonucleotide (RNA or DNA) structure regions that may be important forCRISPR-Cas system function. The structural information provided for Cas9(e.g. S. pyogenes Cas9) as the CRISPR enzyme in the present applicationmay be used to further engineer and optimize the CRISPR-Cas system andthis may be extrapolated to interrogate structure-function relationshipsin other CRISPR enzyme systems as well. The herein cited materialsrelate to the crystal structure of S. pyogenes Cas9 in complex withsgRNA and its target DNA at 2.4 Å resolution. The structure revealed abilobed architecture composed of target recognition and nuclease lobes,accommodating a sgRNA:DNA duplex in a positively-charged groove at theirinterface. The recognition lobe is essential for sgRNA and DNA bindingand the nuclease lobe contains the HNH and RuvC nuclease domains, whichare properly positioned for the cleavage of complementary andnon-complementary strands of the target DNA, respectively. Thishigh-resolution structure and the functional analyses provided hereinelucidate the molecular mechanism of RNA-guided DNA targeting by Cas9,and provides an abundance of information for generating optimizedCRISPR-Cas systems and components thereof. The crystal structure inconjunction with herein teachings may provide steps towardsunderstanding the molecular mechanism of RNA-guided DNA targeting byCas9. The structural and functional analyses of herein teachings andherein cited materials provide a useful scaffold for rationalengineering of Cas9-based genome modulating technologies and may provideguidance as to Cas9-mediated recognition of PAM sequences on the targetDNA or mismatch tolerance between the sgRNA:DNA duplex. Aspects of theinvention may also relate to truncation mutants, e.g. an S. pyogenesCas9 truncation mutant may facilitate packaging of Cas9 intosize-constrained viral vectors for in vivo and therapeutic applications.Similarly, engineering of the PAM Interacting (PI) domain may allowprogramming of PAM specificity, improve target site recognitionfidelity, and increase the versatility of the Cas9 genome engineeringplatform. Cas9 proteins may be engineered to alter their PAMspecificity, for example as described in Kleinstiver B P et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature.2015 Jul. 23; 523(7561):481-5. doi: 10.1038/nature14592.

The invention comprehends optimized functional CRISPR-Cas enzymesystems. In particular the CRISPR enzyme comprises one or more mutationsthat converts it to a DNA binding protein to which ligase domains may berecruited or appended or inserted or attached. In certain embodiments,the CRISPR enzyme comprises one or more mutations which include but arenot limited to D10A, E762A, H840A, N854A, N863A or D986A (based on theamino acid position numbering of a S. pyogenes Cas9) and/or the one ormore mutations is in a RuvC1 or HNH domain of the CRISPR enzyme or is amutation as otherwise as discussed herein. In some embodiments, theCRISPR enzyme has one or more mutations in a catalytic domain, whereinwhen transcribed, the tracr mate sequence hybridizes to the tracrsequence and the guide sequence directs sequence-specific binding of aCRISPR complex to the target sequence.

The teachings herein and structural information of herein citedmaterials provided herein allows for interrogation of sgRNA (or chimericRNA) interaction with the target DNA and the CRISPR enzyme (e.g. Cas9)permitting engineering or alteration of sgRNA structure to optimizefunctionality of the entire CRISPR-Cas system. For example, loops of thesgRNA may be extended, without colliding with the Cas9 protein by theinsertion of distinct RNA loop(s) or distinct sequence(s) that mayrecruit adaptor proteins that can bind to the distinct RNA loop(s) ordistinct sequence(s). The adaptor proteins may include but are notlimited to orthogonal RNA-binding protein/aptamer combinations thatexist within the diversity of bacteriophage coat proteins. A list ofsuch coat proteins includes, but is not limited to: Qβ, F2, GA, fr,JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FL ID2,NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. Theseadaptor proteins or orthogonal RNA binding proteins can further recruiteffector proteins or fusions which comprise one or more ligase domains.

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles. AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356,8,889,418, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233 and8,999,641; US Patent Publication US 2015-0031134 (U.S. application Ser.No. 14/497,627), which is allowed; US Patent Publications US2014-0310830 (U.S. application Ser. No. 14/105,031), US 2014-0287938 A1(U.S. application Ser. No. 14/213,991), US 2014-0273234 A1 (U.S.application Ser. No. 14/293,674), US 2014-0273232 A1 (U.S. applicationSer. No. 14/290,575), US 2014-0273231 (U.S. application Ser. No.14/259,420), US 2014-0256046 A1 (U.S. application Ser. No. 14/226,274),US 2014-0248702 A1 (U.S. application Ser. No. 14/258,458), US2014-0242700 A1 (U.S. application Ser. No. 14/222,930), US 2014-0242699A1 (U.S. application Ser. No. 14/183,512). US 2014-0242664 A1 (U.S.application Ser. No. 14/104,990), US 2014-0234972 A1 (U.S. applicationSer. No. 14/183,471), US 2014-0227787 A1 (U.S. application Ser. No.14/256,912), US 2014-0189896 A1 (U.S. application Ser. No. 14/105,035),US 2014-0186958 (U.S. application Ser. No. 14/105,017), US 2014-0186919A1 (U.S. application Ser. No. 14/104,977). US 2014-0186843 A1 (U.S.application Ser. No. 14/104,900), US 2014-0179770 A1 (U.S. applicationSer. No. 14/104,837) and US 2014-0179006 A1 (U.S. application Ser. No.14/183,486), US 2014-0170753 (U.S. application Ser. No. 14/183,429); US2015-0184139 (U.S. application Ser. No. 14/324,960); Ser. No. 14/054,414European Patent Applications EP 2 771 468 (EP13818570.7). EP 2 764 103(EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT PatentPublications WO 2014/093661 (PCT/US2013/074743), WO 2014/093694(PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611), WO 2014/093718(PCT/US2013/074825), WO 2014/093709 (PCT/US2013/074812). WO 2014/093622(PCT/US2013/074667), WO 2014/093635 (PCT/U S2013/074691), WO 2014/093655(PCT/US2013/074736), WO 2014/093712 (PCT/US2013/074819), WO 2014/093701(PCT/US2013/074800), WO 2014/018423 (PCT/US2013/051418), WO 2014/204723(PCT/US2014/041790). WO 2014/204724 (PCT/US2014/041800), WO 2014/204725(PCT/US2014/041803), WO 2014/204726 (PCT/US2014/041804), WO 2014/204727(PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808), WO 2014/204729(PCT/US2014/041809), WO 2015/089351 (PCT/US2014/069897), WO 2015/089354(PCT/US2014/069902), WO 2015/089364 (PCT/US2014/069925). WO 2015/089427(PCT/US2014/070068), WO 2015/089462 (PCT/US 2014/070127), WO 2015/089419(PCT/US2014/070057), WO 2015/089465 (PCT/US2014/070135), WO 2015/089486(PCT/US2014/070175), PCT/US2015/051691, PCT/US2015/051830. Reference isalso made to U.S. provisional patent applications 61/758,468;61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130, filed onJan. 30, 2013; Mar. 15, 2013; Mar. 28, 2013: Apr. 20, 2013; May 6, 2013and May 28, 2013 respectively. Reference is also made to U.S.provisional patent application 61/836,123, filed on Jun. 17, 2013.Reference is additionally made to U.S. provisional patent applications61/835,931, 61/835,936, 61/835,973, 61/836,080, 61/836,101, and61/836,127, each filed Jun. 17, 2013. Further reference is made to U.S.provisional patent applications 61/862,468 and 61/862,355 filed on Aug.5, 2013; 61/871,301 filed on Aug. 28, 2013; 61/960,777 filed on Sep. 25,2013 and 61/961,980 filed on Oct. 28, 2013. Reference is yet furthermade to: PCT/US2014/62558 filed Oct. 28, 2014, and U.S. ProvisionalPatent Applications Ser. Nos. 61/915,148, 61/915,150, 61/915,153,61/915,203, 61/915,251, 61/915,301, 61/915,267, 61/915,260, and61/915,397, each filed Dec. 12, 2013; 61/757,972 and 61/768,959, filedon Jan. 29, 2013 and Feb. 25, 2013; 62/010,888 and 62/010,879, bothfiled Jun. 11, 2014; 62/010,329, 62/010,439 and 62/010,441, each filedJun. 10, 2014; 61/939,228 and 61/939,242, each filed Feb. 12, 2014;61/980,012, filed Apr. 15, 2014; 62/038,358, filed Aug. 17, 2014;62/055,484, 62/055,460 and 62/055,487, each filed Sep. 25, 2014; and62/069,243, filed Oct. 27, 2014. Reference is made to PCT applicationdesignating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to PCT application designating, inter alia, the UnitedStates, application No. PCT/US14/41806, filed Jun. 10, 2014.

Mention is also made of U.S. application 62/180,709, 17 Jun. 2015,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455, filed, 12Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708,24 Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. applications62/091,462, 12 Dec. 2014, 62/096,324, 23 Dec. 2014, 62/180,681, 17 Jun.2015, and 62/237,496, 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTIONFACTORS: U.S. application 62/091,456, 12 Dec. 2014 and 62/180,692, 17Jun. 2015. ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS;U.S. application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRANDBREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURESEQUENCING; U.S. application 62/096,761, 24 Dec. 2014, ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCEMANIPULATION: U.S. application 62/098,059, 30 Dec. 2014, 62/181,641, 18Jun. 2015, and 62/181,667, 18 Jun. 2015, RNA-TARGETING SYSTEM: U.S.application 62/096,656, 24 Dec. 2014 and 62/181,151, 17 Jun. 2015,CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS; U.S.application 62/096,697, 24 Dec. 2014, CRISPR HAVING OR ASSOCIATED WITHAAV; U.S. application 62/098,158, 30 Dec. 2014, ENGINEERED CRISPRCOMPLEX INSERTIONAL TARGETING SYSTEMS: U.S. application 62/151,052, 22Apr. 2015, CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S.application 62/054,490, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS: USapplication 61/939,154, 12 Feb. 2014, SYSTEMS, METHODS AND COMPOSITIONSFOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/055,484, 25 Sep. 2014, SYSTEMS, METHODS ANDCOMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONALCRISPR-CAS SYSTEMS: U.S. application 62/087,537, 4 Dec. 2014. SYSTEMS,METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/054,651, 24 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCERMUTATIONS IN VIVO; U.S. application 62/067,886, 23 Oct. 2014. DELIVERY,USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS ANDCOMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS INVIVO; U.S. applications 62/054,675, 24 Sep. 2014 and 62/181,002, 17 Jun.2015, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. application62/054,528, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS;U.S. application 62/055,454, 25 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25 Sep. 2014, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 2014 and 62/181,690, 18 Jun. 2015,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/055,487, 25 Sep. 2014, FUNCTIONAL SCREENING WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4Dec. 2014 and 62/181,687, 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES: and U.S.application 62/098,285, 30 Dec. 2014. CRISPR MEDIATED IN VIVO MODELINGAND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Mention is made of U.S. applications 62/181,659, 18 Jun. 2015 and62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FORSEQUENCE MANIPULATION. Mention is made of U.S. applications 62/181,663,18 Jun. 2015 and 62/245,264, 22 Oct. 2015. NOVEL CRISPR ENZYMES ANDSYSTEMS, U.S. applications 62/181,675, 18 Jun. 2015, and Attorney DocketNo. 46783.01.2128, filed 22 Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS,U.S. application 62/232,067, 24 Sep. 2015, U.S. application 62/205,733,16 Aug. 2015, U.S. application 62/201,542, 5 Aug. 2015, U.S. application62/193,507, 16 Jul. 2015, and US application 62/181,739, 18 Jun. 2015,each entitled NOVEL CRISPR ENZYMES AND SYSTEMS and of U.S. application62/245,270, 22 Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Mention isalso made of U.S. application 61/939,256, 12 Feb. 2014, and WO2015/089473 (PCT/US2014/070152), 12 Dec. 2014, each entitled ENGINEERINGOF SYSTEMS. METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEWARCHITECTURES FOR SEQUENCE MANIPULATION. Mention is also made ofPCT/US2015/045504, 15 Aug. 2015, U.S. application 62/180,699, 17 Jun.2015, and U.S. application 62/038,358, 17 Aug. 2014, each entitledGENOME EDITING USING CAS9 NICKASES.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of the following (also hereby incorporated herein by reference):

-   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,    Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D.,    Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February    15; 339(6121):819-23 (2013);-   RNA-guided editing of bacterial genomes using CRISPR-Cas systems.    Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol    March; 31(3):233-9 (2013);-   One-Step Generation of Mice Carrying Mutations in Multiple Genes by    CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila    C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9;    153(4):910-8 (2013);-   Optical control of mammalian endogenous transcription and epigenetic    states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich    M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. 2013    Aug. 22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug.    23;-   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing    Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S.,    Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S.,    Zhang, Y., & Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5.    (2013);-   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,    Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,    Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L    A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);-   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P    D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature    Protocols November; 8(11):2281-308. (2013);-   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem,    O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson,    T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F.    Science December 12. (2013). [Epub ahead of print];-   Crystal structure of cas9 in complex with guide RNA and target DNA.    Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I.,    Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27.    (2014). 156(5):935-49;-   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian    cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D    B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R.,    Zhang F., Sharp P A. Nat Biotechnol. (2014) Apr. 20. doi:    10.1038/nbt.2889,-   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling,    Platt et al., Cell 159(2): 440-455 (2014) DOI:    10.1016/j.cell.2014.09.014,-   Development and Applications of CRISPR-Cas9 for Genome Engineering,    Hsu et al, Cell 157, 1262-1278 (Jun. 5, 2014) (Hsu 2014),-   Genetic screens in human cells using the CRISPR/Cas9 system, Wang et    al., Science. 2014 Jan. 3; 343(6166): 80-84. doi: 10.1126/science.    1246981,-   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated    gene inactivation, Doench et al., Nature Biotechnology published    online 3 Sep. 2014; doi:10.1038/nbt.3026, and-   In vivo interrogation of gene function in the mammalian brain using    CRISPR-Cas9, Swiech et al, Nature Biotechnology; published online 19    Oct. 2014; doi: 10.1038/nbt.3055.-   Genome-scale transcriptional activation by an engineered CRISPR-Cas9    complex, Konermann S. Brigham M D, Trevino A E, Joung J, Abudayyeh O    O, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki    O, Zhang F., Nature. January 29:517(7536):583-8 (2015).-   A split-Cas9 architecture for inducible genome editing and    transcription modulation, Zetsche B, Volz S E, Zhang F., (published    online 2 Feb. 2015) Nat Biotechnol. February; 33(2): 139-42 (2015);-   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and    Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi X,    Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A.    Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in mouse), and-   In vivo genome editing using Staphylococcus aureus Cas9, Ran F A,    Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B,    Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F.,    (published online 1 Apr. 2015), Nature. April 9; 520(7546): 186-91    (2015).-   High-throughput functional genomics using CRISPR-Cas9, Shalem et    al., Nature Reviews Genetics 16, 299-311 (May 2015).-   Sequence determinants of improved CRISPR sgRNA design, Xu et al.,    Genome Research 25, 1147-1157 (August 2015).-   A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect    Regulatory Networks, Parnas et al., Cell 162, 675-686 (Jul. 30,    2015).-   CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B    virus, Ramanan et al., Scientific Reports 5:10833. doi:    10.1038/srep10833 (Jun. 2, 2015).-   Crystal Structure of Staphylococcus aureus Cas9, Nishimasu et al.,    Cell 162, 1113-1126 (Aug. 27, 2015).-   BCL11A enhancer dissection by Cas9-mediated in situ saturating    mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov. 12, 2015)    doi: 10.1038/nature15521. Epub 2015 Sep. 16.-   Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas    System, Zetsche et al., Cell 163, 759-71 (Sep. 25, 2015).-   Discovery and Functional Characterization of Diverse Class 2    CRISPR-Cas Systems, Shmakov et al., Molecular Cell, 60(3), 385-397    doi: 10.1016/j.molcel.2015.10.008 Epub Oct. 22, 2015.-   Rationally engineered Cas9 nucleases with improved specificity,    Slaymaker et al., Science 2015 Dec. 1. pii: aad5227. [Epub ahead of    print]    each of which is incorporated herein by reference, and discussed    briefly below:    -   Cong et al. engineered type II CRISPR/Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptoccocus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR/Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR/Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR/Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. addressed the need in the art for versatile and        robust technologies that enable optical and chemical modulation        of DNA-binding domains based CRISPR Cas9 enzyme and also        Transcriptional Activator Like Effectors.    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays. Shalem et al. (2015) described ways in which        catalytically inactive Cas9 (dCas9) fusions are used to        synthetically repress (CRISPRi) or activate (CRISPRa)        expression, showing. advances using Cas9 for genome-scale        screens, including arrayed and pooled screens, knockout        approaches that inactivate genomic loci and strategies that        modulate transcriptional activity.    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR/Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR/Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional        investigation of non-coding genomic elements. The authors we        developed pooled CRISPR-Cas9 guide RNA libraries to perform in        situ saturating mutagenesis of the human and mouse BCL11A        enhancers which revealed critical features of the enhancers.    -   Zetsche et al. (2015) reported characterization of Cpf1, a class        2 CRISPR nuclease from Francisella novicida U112 having features        distinct from Cas9. Cpf1 is a single RNA-guided endonuclease        lacking tracrRNA, utilizes a T-rich protospacer-adjacent motif,        and cleaves DNA via a staggered DNA double-stranded break.    -   Shmakov et al. (2015) reported three distinct Class 2 CRISPR-Cas        systems. Two system CRISPR enzymes (C2c1 and C2c3) contain        RuvC-like endonuclease domains distantly related to Cpf1. Unlike        Cpf1, C2c1 depends on both crRNA and tracrRNA for DNA cleavage.        The third enzyme (C2c2) contains two predicted HEPN RNase        domains and is tracrRNA independent.    -   Slaymaker et al (2015) reported the use of structure-guided        protein engineering to improve the specificity of Streptococcus        pyogenes Cas9 (SpCas9). The authors developed “enhanced        specificity” SpCas9 (eSpCas9) variants which maintained robust        on-target cleavage with reduced off-target effects.

Mention is also made of Tsai et al, “Dimeric CRISPR RNA-guided Fok1nucleases for highly specific genome editing,” Nature Biotechnology32(6): 569-77 (2014) which is not believed to be prior art to theinstant invention or application, but which may be considered in thepractice of the instant invention. Mention is also made of Konermann etal., “Genome-scale transcription activation by an engineered CRISPR-Cas9complex,” doi:10.1038/nature14136, incorporated herein by reference.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1: Binding of In Vitro Transcribed sgRNA

The inventors assessed the ability of Francisella novicida Cas9 (FnCas9)to bind both native and chimeric RNAs in vitro. It was found thatpurified FnCas9 successfully bound in vitro transcribed scaRNA andtracrRNA, as well as fusion RNA. FnCas9's binding to native mRNA, withand without small RNAs, was also tested, and found that it successfullybound the native FTN_1103 mRNA (FIG. 1).

Example 2: Characterization of Endogenous scaRNA/tracrRNA Transcripts

The CRISPR locus from F. novicida was expressed in E. coli and totalRNA-seq was performed to characterize the endogenous scaRNA and tracrRNAtranscripts. The scaRNA and tracrRNA were present and underwent severalstates of processing (FIG. 2). FIG. 2A provides a graph representing theprocessing states of scaRNA as determined by RNA sequencing (RNA seq).FIG. 2B provides a graph representing the processing states of tracrRNAas determined by RNA sequencing.

Example 3: Analysis of Expression of Exogenous FnCas9 in E. coli

In order to test FnCas9's binding and cleavage in vivo, it wasdetermined that the FnCas9 in the Francisella novicida CRISPR could beexpressed from its native promoter in E. coli. Via Western blot, it wasverified that full-length FnCas9 was expressed successfully (FIG. 3).

Example 4: Reconstitution of FnCas9 mRNA Cleavage in E. coli

After determining that the components of the Francisella novicidaRNA-targeting CRISPR system were successfully expressed in E. coli, itwas assessed that they could cleave the endogenous target, FTN_1103.Exogenous FTN_1103 was introduced into RNA-targeting CRISPR-expressingE. coli strains. Two different variants of FTN_1103, denoted FTN shorttranscript and FTN long transcript, based on the length of the locus,were introduced into RNA-targeting CRISPR-expressing E. coli strains.The amount of cleavage was determined via qPCR. As shown in FIG. 4, theFnCas9 cells have reduced expression of FTN_1103 relative to control (E.coli comprising no FnCas 9 and no F. novicida RNA-targeting CRISPR/Caslocus) indicating knockdown in this system. Next, rather thanintroducing these genes on plasmids, the genes are integrated into theE. coli genome in order to obtain more substantial knockdown consistentwith the natural F. novicida system.

Example 5: Further Characterization of CRISPR/Cas RNA Targeting System

Characterizing Endogenous scaRNA/tracrRNA Transcripts

Since CRISPR RNAs often undergo enzymatic maturation, furtherimmunoprepcipitation and sequencing (IP-seq) of an epitope-tagged FnCas9is performed to reveal the processing state of Fn RNAs in their boundand unbound states (FIG. 5).

Additional Biochemical Characterization

The identified mature Fn RNA sequences are in vitro transcribed andtested with affinity purified FnCas9 protein in gel shift assays, inorder to characterize their biochemical interaction with the endogenouslipoprotein (blp) mRNA target. Different regions of the scaRNA,tracrRNA, and mRNA are mutated to facilitate localization of the guidesequence actually involved in hybridizing with the target lipoproteinmRNA. To gain additional structural insight, FnCas9 in complex with itsguide and target RNAs is crystallized and characterized.

Recognition Rules of FnCas9 to a Target mRNA

FnCas9 recognition of a target mRNA is putatively due to thehybridization between the scaRNA:tracrRNA hybrid (i.e., fSca) and thetarget mRNA, but the recognition pattern is not as straightforward asperfect complementarity. To better understand the binding rules of fScato desired mRNAs, the thermodynamic interaction of candidate guidesequences with target RNAs is computationally modeled to design optimal,retargeted fSca transcripts. In order to understand and refine the rulesunderlying the RNA-targeting FnCas9 complex, a library ofscaRNA/tracrRNA sequences retargeted to eGFP is synthesized. The batteryof guide RNAs is then screened via EMSA, with the fraction bound toFnCas9 and target RNA undergoing reverse transcription, Illumina adapterligation, and deep sequencing (FIG. 6). This high-throughput approachcan reveal the base-pairing rules between the fSca and target mRNA.

Developing a Heterologous RNA Targeting Technology in Human Cells

To test if FnCas9 and fSca can target specific RNAs in mammalian cells,different combinations of the Fn RNA-targeting system are heterologouslyexpressed in HEK293T cells stably expressing eGFP. Byimmunoprecipitating HA-tagged FnCas9 with fSca combinations targetingeGFP transcripts and performing RNA-seq on the RNAs that are pulleddown, binding of the desired target mRNA as well as potential off-targetinteractions are simultaneously tested. qRT-PCR is performed to revealif target eGFP transcripts are degraded, while effects on protein levelsare determined via FACS analysis.

In order to identify any additional protein components of theRNA-targeting FnCas9 regulatory complex, epitope-tagged FnCas9 from F.novicida is immunoprecipitated followed by tandem mass spectrometry(co-IP). The candidate RNases are co-transfected with FnCas9 in HEK 293Tcells and tested for ability to knock down eGFP. If efficient eGFPdegradation is achieved, off-target cleavage and promiscuous RNaseactivity is monitored via whole transcriptome RNA-seq. In case eGFPknockdown fails with the F. novicida-derived RNase, instead a series ofFnCas9 fusions to ribonucleases derived from the metagenomic spectrum isgenerated. This approach can also be used in the event of highoff-target RNA degradation by FnCas9.

To systematically characterize how the fSca guide sequence dictatesFnCas9 specificity and efficiency, a lentiviral-based pooled screen inHEK 293T-GFP cells is developed with two libraries of fSca guidesequences: one on-target library tiling the eGFP locus and oneoff-target library containing only guides with mismatches to the targeteGFP (FIG. 7). FACS-based isolation of eGFP-negative cells followed bydeep sequencing is performed to compare the relative enrichment of guidesequences before and after sorting. More efficient guides or thosetolerating mismatches are expected to be present in higher abundance inthe sorted fraction. This allows investigating the contributions of baseposition and identity for both mismatched and complementary nucleotideson FnCas9 targeting.

Computational Analysis of Cas9 Orthologs for Multiple PutativetracrRNA/scaRNA

Applicants perform an analysis on the Cas9 orthologs from Makarova etal. (Biol. Direct 1, 7 (2006)) to identify multiple putative tracrRNA,implying a system involving a scaRNA, analogous to FnCas9. The Cas9orthologs of interest (attached) are checked for being within 15 kb of apotential CRISPR locus, as determined by the CRISPR recognition tool(Bland, C. et al. BMC Bioinformatics 8, 209 (2007)). All potentialCRISPR loci meeting this criterion are analyzed for sequences within 1kb of the CRISPR locus with 75% complementarity to the direct repeats.Detected sequences above this threshold of complementarity areclassified as putative tracrRNA/scaRNA. Species with more than 1putative tracrRNA/scaRNA are plotted in a phylogenetic tree (FIG. 8). Alist of mined cas9 includes but is not limited to: Campylobacter jejunisubsp. doylei 269.97, Campylobacter jejuni subsp. jejuni 81116,Campylobacter jejuni subsp. jejuni NCTC 11168=ATCC 700819, Kingellakingae ATCC 23330, Gluconacetobacter diazotrophicus PAI 5, Helicobactercanadensis MIT 98-5491, Clostridium cellulolyticum H10, Helicobactercinaedi CCUG 18818, Helicobacter mustelae 12198, gamma proteobacteriumHdN1, Alicycliphilus denitrificans BC, Alicycliphilus denitrificansK601, uncultured Termite group 1 bacterium phylotype Rs-D17, unculturedTermite group 1 bacterium phylotype Rs-D17, Candidatus Puniceispirillummarinum IMCC1322, Parvibaculum lavamentivorans DS-1, Nitrosomonas sp.AL212, Acidovorax avenae subsp. avenae ATCC 19860, Gluconacetobacterdiazotrophicus PAI 5, Aminomonas paucivorans DSM 12260, Haemophilusparainfluenzae T3T1, Staphylococcus aureus subsp. aureus, Actinobacilluspleuropneumoniae serovar 10 str. D13039, Staphylococcus lugdunensisM23590, Pasteurella multocida subsp. multocida str. Pm70, Actinobacillusminor NM305, Wolinella succinogenes DSM 1740, Actinobacillussuccinogenes 130Z, Ralstonia syzygii R24, Rhodopseudomonas palustrisBisB5, Bradyrhizobium sp. BTAi1, Clostridium perfringens D str. JGS1721,Simonsiella muelleri ATCC 29453, Rhodopseudomonas palustris BisB18,Verminephrobacter eiseniae EF01-2, Bacillus cereus Rock1-15, Neisseriabacilliformis ATCC BAA-1200, Dinoroseobacter shibae DFL 12,Methylocystis sp. ATCC 49242, Neisseria flavescens SKI 14, Neisseriameningitidis 053442, Neisseria meningitidis Z2491, Neisseriameningitidis alpha14, Neisseria cinerea ATCC 14685, Methylosinustrichosporium OB3b, Neisseria lactamica 020-06, Corynebacteriumdiphtheriae NCTC 13129, Phascolarctobacterium succinatutens YIT 12067,Corynebacterium matruchotii ATCC 14266, Sphingomonas sp. S17, Mobiluncusmulieris 28-1, Ilyobacter polytropus DSM 2926, Eubacterium dolichum DSM3991, Corynebacterium accolens ATCC 49726, Akkermansia muciniphila ATCCBAA-835, Actinomyces coleocanis DSM 15436, Eubacterium ventriosum ATCC27560, Staphylococcus simulans ACS-120-V-Sch1, Eubacterium rectale ATCC33656, Clostridium spiroforme DSM 1552, Lactobacillus coryniformissubsp. torquens KCTC 3535, Streptococcus thermophilus LMD-9,Streptococcus thermophilus LMG 18311, Streptococcus suis 89/1591,Streptococcus suis ST3, Mobiluncus curtisii subsp. holmesii ATCC 35242,Lactobacillus farciminis KCTC 3681, Streptococcus thermophilus CNRZ1066,Streptococcus vestibularis ATCC 49124, Streptococcus infantarius subsp.infantarius ATCC BAA-102, Streptococcus gallolyticus UCN34,Streptococcus pasteurianus ATCC 43144, Streptococcus macedonicus ACA-DC198, Acidovorax ebreus TPSY, Nitratifractor salsuginis DSM 16511,Streptococcus mitis ATCC 6249, Streptococcus gordonii str. Challissubstr. CH1, Acidothermus cellulolyticus 11B, Bifidobacterium dentiumBd1, Roseburia intestinalis L1-82, Enterococcus faecalis TX0012,Roseburia inulinivorans DSM 16841, Ruminococcus albus 8, Nitrobacterhamburgensis X14, Azospirillum sp. B510, Rhodospirillum rubrum ATCC11170, Scardovia inopinata F0304, Sphaerochaeta globus str. Buddy,Gardnerella vaginalis 5-1, Bifidobacterium longum DJOIOA, Elusimicrobiumminutum Pei 191, Prevotella ruminicola 23, Prevotella buccalis ATCC35310, Prevotella timonensis CRIS 5C-B1, Bacteroides cellulosilyticusDSM 14838, Mycoplasma canis PG 14, Prevotella tannerae ATCC 51259,Mycoplasma mobile 163K, Lactobacillus buchneri ATCC 11577, Mycoplasmaovipneumoniae SC01, Mycoplasma gallisepticum str. F, Mycoplasmagallisepticum str. R(low), Mycoplasma synoviae 53, Streptococcuspseudoporcinus SPIN 20026, Solobacterium moorei F0204, Enterococcusitalicus DSM 15952, Lactobacillus sanfranciscensis TMW 1.1304, Listeriainnocua Clip11262, Listeria monocytogenes str. 1/2a F6854,Staphylococcus pseudintermedius ED99, Coprococcus catus GD/7,Streptococcus macacae NCTC 11558, Dorea longicatena DSM 13814,Enterococcus faecium 1,231,408, Ruminococcus lactaris ATCC 29176,Peptoniphilus sp. oral taxon 386 str. F0131, Streptococcus mutans UA159,Streptococcus mutans NN2025, Streptococcus anginosus F0211, Finegoldiamagna ATCC 29328, Streptococcus equi subsp. zooepidemicus MGCS10565,Megasphaera sp. UPII 135-E, Flavobacterium psychrophilum JIP02/86,Prevotella melaninogenica D18, Acidaminococcus sp. D21, Acidaminococcusintestini RyC-MR95, Lactobacillus casei BL23, Anaerococcus tetradiusATCC 35098, Lactobacillus casei str. Zhang, Lactobacillus paracaseisubsp. paracasei 8700:2, Lactobacillus rhamnosus GG, Pediococcusacidilactici DSM 20284, Peptoniphilus duerdenii ATCC BAA-1640,Filifactor alocis ATCC 35896, Streptococcus pyogenes M1 GAS,Streptococcus pyogenes MGAS315, Streptococcus pyogenes SSI-1,Streptococcus pyogenes MGAS6180, Streptococcus pyogenes MGAS5005,Streptococcus pyogenes MGAS9429, Streptococcus pyogenes MGAS10270,Streptococcus pyogenes MGAS2096, Streptococcus pyogenes NZ131,Lactobacillus iners LactinV 11V1-d, Streptococcus agalactiae 2603V/R,Streptococcus agalactiae A909, Streptococcus gallolyticus subsp.gallolyticus ATCC BAA-2069, Streptococcus pyogenes MGAS10750,Streptococcus dysgalactiae subsp. equisimilis GGS_124, Streptococcusgallolyticus UCN34, Gordonibacter pamelaeae 7-10-1-b, Lactobacillusbuchneri NRRL B-30929, Legionella pneumophila str. Paris, Streptococcusbovis ATCC 700338, Fusobacterium nucleatum subsp. vincentii ATCC 49256,Lactobacillus ruminis ATCC 25644, Lactobacillus johnsonii DPC 6026,Streptococcus agalactiae NEM316, Lactobacillus brevis subsp. gravesensisATCC 27305, Streptococcus equinus ATCC 9812, Eggerthella sp. YY7918,Lactobacillus fermentum ATCC 14931, Coriobacterium glomerans PW2,Gemella morbillorum M424, Streptococcus thermophilus LMD-9, Zunongwangiaprofunda SM-A87, Oenococcus kitaharae DSM 17330, Kordia algicida OT-1,Lactobacillus jensenii 269-3, Lactobacillus gasseri JV-V03, Prevotellaoralis ATCC 33269, Gemella haemolysans ATCC 10379, Treponema denticolaATCC 35405, gamma proteobacterium HTCC5015, Veillonella parvula ATCC17745, Veillonella atypica ACS-134-V-Col7a, Olsenella uli DSM 7084,Wolinella succinogenes DSM 1740, Bifidobacterium bifidum S17, Sutterellawadsworthensis 3_1_45B, Parabacteroides sp. D13, Prevotella micansF0438, Capnocytophaga sputigena Capno, Capnocytophaga ochracea DSM 7271,Sphingobacterium spiritivorum ATCC 33861, Burkholderiales bacterium1_1_47, Parasutterella excrementihominis YIT 11859, Capnocytophagacanimorsus Cc5, Bacteroidetes oral taxon 274 str. F0058, Bacteroidesfragilis NCTC 9343, Capnocytophaga gingivalis ATCC 33624, Weeksellavirosa DSM 16922, Parabacteroides johnsonii DSM 18315, Prevotella buccaeATCC 33574, Fluviicola taffensis DSM 16823, Flavobacterium columnareATCC 49512, Flavobacterium branchiophilum FL-15, Mucilaginibacterpaludis DSM 18603, Prevotella bivia JCVIHMP010, Prevotella veroralisF0319, Bacteroides dorei DSM 17855, Bacteroides coprophilus DSM 18228,Fibrobacter succinogenes subsp. succinogenes S85, Bacteroides sp. 20_3,Flavobacterium columnare ATCC 49512, and Francisella novicida U112.

In the event that FnCas9 expresses or functions poorly in eukaryoticcells, metagenomic discovery is performed to mine alternative Cas9s thatcould interact with RNA. Several CRISPR systems have been implicated ingene regulatory functions at the RNA level, and computational analysisreveals many type II CRISPR loci that possess multiple putativetranscripts possessing anti-direct repeat sequences. The subset of these(50% or less) that do not serve as canonical tracrRNAs for crRNAtargeting may be prokaryotic small RNAs involved in post-transcriptionalregulation. A combination of RNA-seq and Cas9 cross-linkingimmunoprecipitation sequencing (CLIP-Seq) in these prokaryotesfacilitates further functional characterization.

Example 6: scaRNA

FIG. 9A provides the sequence of scaRNA from Sampson et al. (2013,Nature 497: 254-258).

FIG. 9B provides the sequence of Applicants' scaRNA from RNA sequencingand FIG. 9C provides RNA Seq reads on ScaRNA.

The invention is further described by the following numbered paragraphs:

-   -   1. A non-naturally occurring or engineered vector system        comprising one or more vectors comprising:

a) a first regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting guide RNA (Rt-gRNA), wherein saidRNA-targeting guide RNA is capable of hybridizing with a target RNA,wherein said RNA-targeting guide RNA comprises:

(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and

(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein said scaRNA and said tracrRNA are capable of at least partiallyhybridizing, and

b) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein,

wherein components (a) and (b) are located on the same or differentvectors of the system.

-   -   2. The vector system according to numbered paragraph 1, wherein        said RNA-targeting guide RNA and said RNA-targeting Cas protein        do not naturally occur together.    -   3. The vector system according to numbered paragraph 1 or 2,        wherein said RNA-targeting Cas protein is a type II Cas protein.    -   4. The vector system according to numbered paragraph 3, wherein        said RNA-targeting Cas protein is a Cas9 protein.    -   5. The vector system according to claim 3, wherein said        RNA-targeting Cas protein is from Francisella novicida.    -   6. The vector system according to numbered paragraph 3, wherein        said RNA-targeting Cas protein is the Cas9 protein from        Francisella novicida (FnCas9).    -   7. The vector system according to numbered paragraph 3, wherein        said RNA-targeting Cas protein is an FnCas9 homolog showing at        least 80% sequence homology with wild type FnCas9.    -   8. The vector system according to numbered paragraph 3, wherein        said RNA-targeting Cas protein is an FnCas9 ortholog.    -   9. The vector system according to numbered paragraph 1, wherein        said scaRNA sequence is fused to said tracrRNA sequence.    -   10. The vector system according to numbered paragraph 1, wherein        said RNA-targeting Cas protein is codon optimized for expression        in a eukaryotic cell.    -   11. The vector system according to numbered paragraph 1, wherein        said one or more vectors are viral vectors.    -   12. The vector system according to numbered paragraph 1, wherein        said one or more viral vectors are selected from the group        consisting of retroviral, lentiviral, adenoviral,        adeno-associated and herpes simplex viral vectors.    -   13. The vector system according to numbered paragraph 1, wherein        said RNA-targeting guide RNA is capable of directing said        RNA-targeting Cas protein to said target RNA.    -   14. A vector comprising a regulatory element operably linked to        a polynucleotide sequence encoding an RNA-targeting Cas protein,        preferably FnCas9 or an FnCas9 homolog or ortholog.    -   15. A vector comprising a regulatory element operably linked to        a polynucleotide sequence encoding an RNA-targeting guide RNA,        wherein said RNA-targeting guide RNA is capable of hybridizing        with a target RNA, wherein said RNA-targeting guide RNA        comprises:

(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and

(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein said scaRNA and said tracrRNA are capable of at least partiallyhybridizing.

-   -   16. A composition comprising a vector system according to        numbered paragraph 1 or a vector according to numbered paragraph        14 or 15.    -   17. A non-naturally occurring or engineered RNA-targeting system        comprising an RNA-targeting Cas protein and at least one        RNA-targeting guide RNA, wherein said RNA-targeting guide RNA is        capable of binding to a target RNA in an eukaryotic cell,        wherein said RNA-targeting guide RNA comprises:

(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and

(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein said scaRNA and said tracrRNA are capable of at least partiallyhybridizing.

-   -   18. The system according to numbered paragraph 17, wherein said        RNA-targeting Cas protein is FnCas9 or an FnCas9 homolog or        ortholog.    -   19. A method for modulating synthesis of a protein comprising        introducing into a cell containing an RNA molecule encoding said        protein, a vector system comprising one or more vectors        comprising:

a) a first regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting guide RNA, wherein said RNA-targetingguide RNA is capable of hybridizing with said target RNA, wherein saidRNA-targeting guide RNA comprises:

(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and

(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein said scaRNA and said tracrRNA are capable of at least partiallyhybridizing, and

b) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein,

wherein components (a) and (b) are located on the same or differentvectors of the system,

whereby synthesis of said protein is modulated.

-   -   20. The method according to numbered paragraph 19, wherein the        synthesis of said protein is suppressed.    -   21. The method according to numbered paragraph 20, wherein the        synthesis of said protein is suppressed by knock-down of said        RNA molecule encoding said protein.    -   22. The method according to numbered paragraph 19, wherein the        synthesis of said protein is modulated by editing of said RNA        molecule encoding said protein.    -   23. The method according to numbered paragraph 15, wherein the        synthesis of said protein is modulated by splicing of said RNA        molecule encoding said protein.    -   24. A method for targeting RNA comprising introducing into a        cell containing a target RNA a non-naturally occurring or        engineered vector system comprising one or more vectors        comprising:

a) a first regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting guide RNA, wherein said RNA-targetingguide RNA is capable of hybridizing with said target RNA, wherein saidRNA-targeting guide RNA comprises:

(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and

(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence,wherein said scaRNA and said tracrRNA are capable of at least partiallyhybridizing, and

b) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein,

wherein components (a) and (b) are located on the same or differentvectors of the system,

whereby said RNA-targeting guide RNA directs said RNA-targeting Casprotein to said target RNA.

-   -   25. The method according to numbered paragraph 19 or 24, wherein        said RNA-targeting Cas protein is a type II Cas protein.    -   26. The method according to numbered paragraph 25 wherein said        RNA-targeting Cas protein is a Cas9 protein.    -   27. The method according to numbered paragraph 25, wherein said        RNA-targeting Cas protein is from Francisella novicida.    -   28. The method according to numbered paragraph 25, wherein said        RNA-targeting Cas protein is an FnCas9 homolog showing at least        80% sequence homology with wild type FnCas9.    -   29. The method according to numbered paragraph 25, wherein said        RNA-targeting Cas protein is an FnCas9 ortholog.    -   30. The method according to numbered paragraph 19 or 24, wherein        said RNA-targeting Cas protein is codon optimized for expression        in a eukaryotic cell.    -   31. The method according to numbered paragraph 19 or 24, wherein        said one or more vectors are viral vectors.    -   32. The method according to numbered paragraph 19 or 24, wherein        said one or more viral vectors are selected from the group        consisting of retroviral, lentiviral, adenoviral,        adeno-associated and herpes simplex viral vectors.    -   33. The method according to numbered paragraph 19 or 24, wherein        the cell is a eukaryotic cell.    -   34. The method according to numbered paragraph 30, wherein the        eukaryotic cell is a mammalian or human cell.    -   35. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, wherein one or        more amino acid residues of the RNA-targeting Cas protein are        modified.    -   36. The vector system, vector, composition or method according        to numbered paragraph 35, wherein the modification comprises        mutation of one or more, or two or more, or three or more amino        acid residues of the RNA-targeting Cas protein.    -   37. The vector system, vector, composition or method according        to numbered paragraph 35 or 36, wherein the one or more, or two        or more, or three or more mutations are in one or more        catalytically active domains of the RNA-targeting Cas protein.    -   38. The vector system, vector, composition or method according        to numbered paragraph 37, wherein the RNA-targeting Cas protein        has reduced or abolished nuclease activity compared with an        RNA-targeting Cas protein lacking said mutation(s).    -   39. The vector system, vector, composition or method according        to numbered paragraph 38, wherein the one or more, or two or        more mutations are in a catalytically active domain of the        RNA-targeting Cas protein comprising a RuvCI, RuvCII or RuvCIII        domain.    -   40. The vector system, vector, composition or method according        to any one of numbered paragraphs 35 to 39, wherein the        RNA-targeting Cas protein comprises one or more heterologous        functional domains.    -   41. The vector system, vector, composition or method according        to numbered paragraph 40, wherein the one or more heterologous        functional domains comprises one or more nuclear localization        signal (NLS) domains.    -   42. The vector system, vector, composition or method according        to numbered paragraph 41, wherein the one or more heterologous        functional domains comprises at least two or more NLS domains.    -   43. The vector system, vector, composition or method according        to numbered paragraph 40, wherein the one or more heterologous        functional domains comprises one or more transcriptional        activation domains.    -   44. The vector system, vector, composition or method according        to numbered paragraph 43, wherein the transcriptional activation        domain comprises VP64.    -   45. The vector system, vector, composition or method according        to numbered paragraph 40, wherein the one or more heterologous        functional domains comprises one or more transcriptional        repression domains.    -   46. The vector system, vector, composition or method according        to numbered paragraph 45, wherein the transcriptional repression        domain comprises a KRAB domain or a SID domain.    -   47. The vector system, vector, composition or method according        to numbered paragraph 40, wherein the one or more heterologous        functional domains comprises one or more nuclease domains.    -   48. The vector system, vector, composition or method according        to numbered paragraph 47, wherein a nuclease domain comprises        Fok1.    -   49. The vector system, vector, composition or method according        to numbered paragraph 40, wherein the one or more heterologous        functional domains have one or more of the following activities:        methylase activity, demethylase activity, transcription        activation activity, transcription repression activity,        transcription release factor activity, histone modification        activity, nuclease activity, single-strand RNA cleavage        activity, double-strand RNA cleavage activity, single-strand DNA        cleavage activity, double-strand DNA cleavage activity and        nucleic acid binding activity.    -   50. The vector system, vector, composition or method according        to any one of numbered paragraphs 40 to 49, wherein at least one        or more heterologous functional domains is at or near the        amino-terminus of the RNA-targeting Cas protein and/or wherein        at least one or more heterologous functional domains is at or        near the carboxy-terminus of the RNA-targeting Cas protein.    -   51. The vector system, vector, composition or method according        to any one of numbered paragraphs 40 to 50, wherein said one or        more heterologous functional domains are fused to the        RNA-targeting Cas protein.    -   52. The vector system, vector, composition or method according        to any one of numbered paragraphs 40 to 50, wherein said one or        more heterologous functional domains are tethered to the        RNA-targeting Cas protein.    -   53. The vector system, vector, composition or method according        to any one of numbered paragraphs 40 to 50, wherein said one or        more heterologous functional domains are linked to the        RNA-targeting Cas protein by a linker moiety.    -   54. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, wherein the        RNA-targeting Cas protein comprises an RNA-targeting Cas protein        from an organism from a genus comprising Streptococcus,        Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum,        Roseburia, Neisseria, Gluconacetobacter, Azospirillum,        Sphaerochaeta, Lactobacillus, Eubacterium or Corynebacter.    -   55. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, wherein the        RNA-targeting Cas protein comprises a chimeric RNA-targeting Cas        protein comprising a first fragment from a first RNA-targeting        Cas protein ortholog and a second fragment from a second        RNA-targeting Cas protein ortholog, and wherein the first and        second RNA-targeting Cas protein orthologs are different,        wherein at least one of the first and second orthologs is        Francisella novicida.    -   56. The vector system, vector, composition or method according        to numbered paragraph 55, wherein at least one of the first and        second RNA-targeting Cas protein orthologs comprises an        RNA-targeting Cas protein from an organism comprising        Streptococcus, Campylobacter, Nitratifractor, Staphylococcus,        Parvibaculum, Roseburia, Neisseria, Gluconacetobacter,        Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium or        Corynebacter.    -   57. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, wherein the        tracrRNA comprises one or more protein-binding RNA aptamers.    -   58. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, wherein the        scaRNA comprises one or more protein-binding RNA aptamers.    -   59. The vector system, vector, composition or method according        to numbered paragraph 57 or numbered paragraph 58, wherein the        one or more aptamers is capable of binding a bacteriophage coat        protein.    -   60. The vector system, vector, composition or method according        to numbered paragraph 59, wherein the bacteriophage coat protein        is selected from the group comprising Qβ, F2, GA, fr, JP501,        MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP,        FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and        PRR1.    -   61. The vector system, vector, composition or method according        to numbered paragraph 60, wherein the bacteriophage coat protein        is MS2.    -   62. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, wherein the        tracrRNA is 30 or more, 40 or more or 50 or more nucleotides in        length.    -   63. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, wherein the        RNA-targeting Cas protein is further modified to comprise PAM        sequence specificity which is different to the PAM sequence        specificity of the RNA-targeting Cas protein without said        further modification.    -   64. The vector system, vector, composition or method according        to numbered paragraph 63, wherein said further modification        comprises the introduction of one or more amino acid mutations        into the RNA-targeting Cas protein, or by truncation of the        RNA-targeting Cas protein, or by deletion and/or insertion of        specific amino acids or amino acid sequences into the        RNA-targeting Cas protein.    -   65. The vector system, vector, composition or method according        to any one of the preceding numbered paragraphs, comprising        delivering multiple Rt-gRNAs, wherein each Rt-gRNAs is specific        for a different target RNA whereby there is multiplexing.    -   66. A host cell or cell line comprising the vector system,        vector, or composition according to any one of numbered        paragraphs 1-18 or 35-65, or progeny thereof.    -   67. The host cell or cell line or progeny thereof according to        numbered paragraph 66, which is ex vivo or in vitro.    -   68. The host cell or cell line or progeny thereof according to        any one of numbered paragraphs 66-67, which is a stem cell or a        stem cell line, or a plant cell or a plant cell line.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed:
 1. A non-naturally occurring or engineered vectorsystem comprising one or more vectors comprising: a) a first regulatoryelement operably linked to a polynucleotide sequence encoding anRNA-targeting guide RNA (Rt-gRNA), wherein said RNA-targeting guide RNAis capable of hybridizing with a target RNA, wherein said RNA-targetingguide RNA comprises: (i) a small CRISPR/Cas system associated RNA(scaRNA) sequence, and (ii) a trans-activating CRISPR/Cas system RNA(tracrRNA) sequence, wherein said scaRNA and said tracrRNA are capableof at least partially hybridizing, and b) a second regulatory elementoperably linked to a polynucleotide sequence encoding an RNA-targetingCas protein, wherein components (a) and (b) are located on the same ordifferent vectors of the system.
 2. The vector system according to claim1, wherein said RNA-targeting guide RNA and said RNA-targeting Casprotein do not naturally occur together.
 3. The vector system accordingto claim 1, wherein said RNA-targeting Cas protein is a type II Casprotein.
 4. The vector system according to claim 3, wherein saidRNA-targeting Cas protein is a Cas9 protein.
 5. The vector systemaccording to claim 3, wherein said RNA-targeting Cas protein is fromFrancisella novicida.
 6. The vector system according to claim 3, whereinsaid RNA-targeting Cas protein is the Cas9 protein from Francisellanovicida (FnCas9).
 7. The vector system according to claim 3, whereinsaid RNA-targeting Cas protein is an FnCas9 homolog showing at least 80%sequence homology with wild type FnCas9.
 8. The vector system accordingto claim 3, wherein said RNA-targeting Cas protein is an FnCas9ortholog.
 9. The vector system according to claim 1, wherein said scaRNAsequence is fused to said tracrRNA sequence.
 10. The vector systemaccording to claim 1, wherein said RNA-targeting Cas protein is codonoptimized for expression in a eukaryotic cell.
 11. The vector systemaccording to claim 1, wherein said one or more vectors are viralvectors.
 12. The vector system according to claim 1, wherein said one ormore viral vectors are selected from the group consisting of retroviral,lentiviral, adenoviral, adeno-associated and herpes simplex viralvectors.
 13. The vector system according to claim 1, wherein saidRNA-targeting guide RNA is capable of directing said RNA-targeting Casprotein to said target RNA.
 14. A vector comprising a regulatory elementoperably linked to a polynucleotide sequence encoding an RNA-targetingCas protein, preferably FnCas9 or an FnCas9 homolog or ortholog.
 15. Avector comprising a regulatory element operably linked to apolynucleotide sequence encoding an RNA-targeting guide RNA, whereinsaid RNA-targeting guide RNA is capable of hybridizing with a targetRNA, wherein said RNA-targeting guide RNA comprises: (i) a smallCRISPR/Cas system associated RNA (scaRNA) sequence, and (ii) atrans-activating CRISPR/Cas system RNA (tracrRNA) sequence, wherein saidscaRNA and said tracrRNA are capable of at least partially hybridizing.16. A composition comprising a vector system according to claim 1 or avector according to claim
 14. 17. A non-naturally occurring orengineered RNA-targeting system comprising an RNA-targeting Cas proteinand at least one RNA-targeting guide RNA, wherein said RNA-targetingguide RNA is capable of binding to a target RNA in an eukaryotic cell,wherein said RNA-targeting guide RNA comprises: (i) a small CRISPR/Cassystem associated RNA (scaRNA) sequence, and (ii) a trans-activatingCRISPR/Cas system RNA (tracrRNA) sequence, wherein said scaRNA and saidtracrRNA are capable of at least partially hybridizing.
 18. The systemaccording to claim 17, wherein said RNA-targeting Cas protein is FnCas9or an FnCas9 homolog or ortholog.
 19. A method for modulating synthesisof a protein comprising introducing into a cell containing an RNAmolecule encoding said protein, a vector system comprising one or morevectors comprising: a) a first regulatory element operably linked to apolynucleotide sequence encoding an RNA-targeting guide RNA, whereinsaid RNA-targeting guide RNA is capable of hybridizing with said targetRNA, wherein said RNA-targeting guide RNA comprises: (i) a smallCRISPR/Cas system associated RNA (scaRNA) sequence, and (ii) atrans-activating CRISPR/Cas system RNA (tracrRNA) sequence, wherein saidscaRNA and said tracrRNA are capable of at least partially hybridizing,and b) a second regulatory element operably linked to a polynucleotidesequence encoding an RNA-targeting Cas protein, wherein components (a)and (b) are located on the same or different vectors of the system,whereby synthesis of said protein is modulated.
 20. The method accordingto claim 19, wherein the synthesis of said protein is suppressed. 21.The method according to claim 20, wherein the synthesis of said proteinis suppressed by knock-down of said RNA molecule encoding said protein.22. The method according to claim 19, wherein the synthesis of saidprotein is modulated by editing of said RNA molecule encoding saidprotein.
 23. The method according to claim 15, wherein the synthesis ofsaid protein is modulated by splicing of said RNA molecule encoding saidprotein.
 24. A method for targeting RNA comprising introducing into acell containing a target RNA a non-naturally occurring or engineeredvector system comprising one or more vectors comprising: a) a firstregulatory element operably linked to a polynucleotide sequence encodingan RNA-targeting guide RNA, wherein said RNA-targeting guide RNA iscapable of hybridizing with said target RNA, wherein said RNA-targetingguide RNA comprises: (i) a small CRISPR/Cas system associated RNA(scaRNA) sequence, and (ii) a trans-activating CRISPR/Cas system RNA(tracrRNA) sequence, wherein said scaRNA and said tracrRNA are capableof at least partially hybridizing, and b) a second regulatory elementoperably linked to a polynucleotide sequence encoding an RNA-targetingCas protein, wherein components (a) and (b) are located on the same ordifferent vectors of the system, whereby said RNA-targeting guide RNAdirects said RNA-targeting Cas protein to said target RNA.
 25. Themethod according to claim 19, wherein said RNA-targeting Cas protein isa type II Cas protein.
 26. The method according to claim 25, whereinsaid RNA-targeting Cas protein is a Cas9 protein.
 27. The methodaccording to claim 25, wherein said RNA-targeting Cas protein is fromFrancisella novicida.
 28. The method according to claim 25, wherein saidRNA-targeting Cas protein is an FnCas9 homolog showing at least 80%sequence homology with wild type FnCas9.
 29. The method according toclaim 25, wherein said RNA-targeting Cas protein is an FnCas9 ortholog.30. The method according to claim 19, wherein said RNA-targeting Casprotein is codon optimized for expression in a eukaryotic cell.
 31. Themethod according to claim 19, wherein said one or more vectors are viralvectors.
 32. The method according to claim 19, wherein said one or moreviral vectors are selected from the group consisting of retroviral,lentiviral, adenoviral, adeno-associated and herpes simplex viralvectors.
 33. The method according to claim 19, wherein the cell is aeukaryotic cell.
 34. The method according to claim 30, wherein theeukaryotic cell is a mammalian or human cell.
 35. The vector system,vector, composition or method according to claim, wherein one or moreamino acid residues of the RNA-targeting Cas protein are modified. 36.The vector system, vector, composition or method according to claim 35,wherein the modification comprises mutation of one or more, or two ormore, or three or more amino acid residues of the RNA-targeting Casprotein.
 37. The vector system, vector, composition or method accordingto claim 35, wherein the one or more, or two or more, or three or moremutations are in one or more catalytically active domains of theRNA-targeting Cas protein.
 38. The vector system, vector, composition ormethod according to claim 37, wherein the RNA-targeting Cas protein hasreduced or abolished nuclease activity compared with an RNA-targetingCas protein lacking said mutation(s).
 39. The vector system, vector,composition or method according to claim 38, wherein the one or more, ortwo or more mutations are in a catalytically active domain of theRNA-targeting Cas protein comprising a RuvCI, RuvCII or RuvCIII domain.40. The vector system, vector, composition or method according to claim35, wherein the RNA-targeting Cas protein comprises one or moreheterologous functional domains.
 41. The vector system, vector,composition or method according to claim 40, wherein the one or moreheterologous functional domains comprises one or more nuclearlocalization signal (NLS) domains.
 42. The vector system, vector,composition or method according to claim 41, wherein the one or moreheterologous functional domains comprises at least two or more NLSdomains.
 43. The vector system, vector, composition or method accordingto claim 40, wherein the one or more heterologous functional domainscomprises one or more transcriptional activation domains.
 44. The vectorsystem, vector, composition or method according to claim 43, wherein thetranscriptional activation domain comprises VP64.
 45. The vector system,vector, composition or method according to claim 40, wherein the one ormore heterologous functional domains comprises one or moretranscriptional repression domains.
 46. The vector system, vector,composition or method according to claim 45, wherein the transcriptionalrepression domain comprises a KRAB domain or a SID domain.
 47. Thevector system, vector, composition or method according to claim 40,wherein the one or more heterologous functional domains comprises one ormore nuclease domains.
 48. The vector system, vector, composition ormethod according to claim 47, wherein a nuclease domain comprises Fok1.49. The vector system, vector, composition or method according to claim40, wherein the one or more heterologous functional domains have one ormore of the following activities: methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, nuclease activity, single-strand RNA cleavage activity,double-strand RNA cleavage activity, single-strand DNA cleavageactivity, double-strand DNA cleavage activity and nucleic acid bindingactivity.
 50. The vector system, vector, composition or method accordingto claim 40, wherein at least one or more heterologous functionaldomains is at or near the amino-terminus of the RNA-targeting Casprotein and/or wherein at least one or more heterologous functionaldomains is at or near the carboxy-terminus of the RNA-targeting Casprotein.
 51. The vector system, vector, composition or method accordingto claim 40, wherein said one or more heterologous functional domainsare fused to the RNA-targeting Cas protein.
 52. The vector system,vector, composition or method according to claim 40, wherein said one ormore heterologous functional domains are tethered to the RNA-targetingCas protein.
 53. The vector system, vector, composition or methodaccording to claim 40, wherein said one or more heterologous functionaldomains are linked to the RNA-targeting Cas protein by a linker moiety.54. The vector system, vector, composition or method according to claim,wherein the RNA-targeting Cas protein comprises an RNA-targeting Casprotein from an organism from a genus comprising Streptococcus,Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta,Lactobacillus, Eubacterium or Corynebacter.
 55. The vector system,vector, composition or method according to claim, wherein theRNA-targeting Cas protein comprises a chimeric RNA-targeting Cas proteincomprising a first fragment from a first RNA-targeting Cas proteinortholog and a second fragment from a second RNA-targeting Cas proteinortholog, and wherein the first and second RNA-targeting Cas proteinorthologs are different, wherein at least one of the first and secondorthologs is Francisella novicida.
 56. The vector system, vector,composition or method according to claim 55, wherein at least one of thefirst and second RNA-targeting Cas protein orthologs comprises anRNA-targeting Cas protein from an organism comprising Streptococcus,Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta,Lactobacillus, Eubacterium or Corynebacter.
 57. The vector system,vector, composition or method according to claim, wherein the tracrRNAcomprises one or more protein-binding RNA aptamers.
 58. The vectorsystem, vector, composition or method according to claim, wherein thescaRNA comprises one or more protein-binding RNA aptamers.
 59. Thevector system, vector, composition or method according to claim 57,wherein the one or more aptamers is capable of binding a bacteriophagecoat protein.
 60. The vector system, vector, composition or methodaccording to claim 59, wherein the bacteriophage coat protein isselected from the group comprising Qβ, F2, GA, fr, JP501, MS2, M12, R17,BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19,AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1.
 61. The vector system,vector, composition or method according to claim 60, wherein thebacteriophage coat protein is MS2.
 62. The vector system, vector,composition or method according to claim, wherein the tracrRNA is 30 ormore, 40 or more or 50 or more nucleotides in length.
 63. The vectorsystem, vector, composition or method according to claim, wherein theRNA-targeting Cas protein is further modified to comprise PAM sequencespecificity which is different to the PAM sequence specificity of theRNA-targeting Cas protein without said further modification.
 64. Thevector system, vector, composition or method according to claim 63,wherein said further modification comprises the introduction of one ormore amino acid mutations into the RNA-targeting Cas protein, or bytruncation of the RNA-targeting Cas protein, or by deletion and/orinsertion of specific amino acids or amino acid sequences into theRNA-targeting Cas protein.
 65. The vector system, vector, composition ormethod according to claim, comprising delivering multiple Rt-gRNAs,wherein each Rt-gRNAs is specific for a different target RNA wherebythere is multiplexing.
 66. A host cell or cell line comprising thevector system, vector, or composition according to claim 1, or progenythereof.
 67. The host cell or cell line or progeny thereof according toclaim 66, which is ex vivo or in vitro.
 68. The host cell or cell lineor progeny thereof according to claim 66, which is a stem cell or a stemcell line, or a plant cell or a plant cell line.